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Closed reduction is applied in most cases of isolated zygomatic arch fracture. Open reduction and internal rigid fixation through a coronal incision have to be performed in other comminuted arch fractures. The authors introduced an endoscopic-assisted approach via a small preauricular incision to achieve reposition and osteosynthesis of isolated zygomatic arch fractures. The endoscopic-assisted surgical technique was performed in 7 patients with unilateral isolated zygomatic arch fractures. Endoscopically controlled management of the isolated zygomatic arch fracture was feasible in all 7 patients. The follow-up period was 1 year. All preauricular scar and facial lateral contour were aesthetically satisfactory. The endoscope-assisted approach via a small preauricular incision can achieve in situ reduction and fixation in zygomatic arch fracture and it should become an integral part of isolated zygomatic fracture repair, assuming the development of specialized training programs and improvements in endoscopes.

The potential impact of the surgical correction of strabismus on vision-related quality of life (VRQOL) and the symptoms of anxiety and depression in children with strabismus remain unclear. The present study included 60 children with strabismus: 30 with heterophoria and 30 with heterotropia. A healthy age- and gender-matched control group (n = 60) was also recruited. The psychological instruments that were used were the short-form 25-item National Eye Institute Visual Functioning Questionnaire (NEI-VFQ-25) and the Hospital Anxiety and Depression Scale (HADS). The results demonstrated that eight of the 12 NEI-VFQ-25 subscales were significantly impaired in children with strabismus compared with matched controls. Compared with pre-operative values, significant improvements were noted after surgery in the NEI-VFQ-25 summary score, and the anxiety and depression scores. This study demonstrated that the NEI-VFQ-25 instrument can be used in strabismus children and that surgical interventions can improve VRQOL, anxiety and depression in strabismus patients.

The experiment was conducted to evaluate the effect of dietary supplementation with 0, 50, 100 and 200 mg/kg of beta-1,3/1,6-glucan extracted from Saccharomyces cerevisiae on cellular immune responses and classical swine fever virus (CSFV) antibody production of weaned piglets. Thirty-two (Landrace x Large White; 26 days old; 7.05+/-0.25 kg body weight) castrated male piglets were randomly allocated to four treatments, eight pigs per treatment. All piglets were vaccinated with live-attenuated Chinese strain cell vaccines at 26 days of age. The results showed that dietary supplementation with beta-1,3/1,6-glucan could significantly (P<0.01) enhance peripheral blood lymphocyte proliferation activity in weaned piglets after 14 days administration, whereas no significant effect (P>0.05) was observed on day 28. Dietary beta-1,3/1,6-glucan supplementation could significantly enhance CSFV antibody blocking rate (P<0.05) on days 14 and 21 after inoculation. Although dietary beta-1,3/1,6-glucan supplementation did not significantly influence (P>0.05) lymphocyte subgroups on day 14, high levels of beta-1,3/1,6-glucan (100 and 200 mg/kg) increased the percentage of cytotoxic/suppressor lymphocytes (CD8(+)) significantly (P<0.05), decreased the ratio of CD4(+)/CD8(+) significantly (P<0.05) and increased the percentage of total T lymphocytes (CD3(+)) on day 28. These results suggest that beta-1,3/1,6-glucan may have a beneficial effect on the immune system of swine, and dietary beta-1,3/1,6-glucan supplementation had a transient impact on lymphocyte proliferation activity. The CSFV antibody blocking rate and the percentage of peripheral blood CD8(+) T cells could be enhanced by continuous beta-1,3/1,6-glucan supplementation. These findings support the potential application of beta-1,3/1,6-glucan as a prophylactic agent in increasing animal immune functions and controlling CSF disease.

Five ORFs in the genome of Duck enteritis virus (DEV) corresponding to UL31, UL32, UL33, UL34, and UL35 genes of Herpes simplex virus 1 (HSV-1) were amplified by a modified "targeted gene walking" PCR, cloned, and sequenced. UL33, UL34, and UL35 genes were oriented from the left to the right of genome, while UL31 and UL32 had an opposite orientation. A comparison of deduced amino acid sequences of the DEV ORFs with their alphaherpesvirus homologs showed well-conserved regions except for the UL34 and UL35 genes. Phylogenetic analysis revealed that DEV was closer to the genus Mardivirus than to any other genus of the subfamily Alphaherpesvirinae. Based on this evidence, we proposed to assign DEV to the subfamily Alphaherpesvirinae.

The aims of this study were to investigate whether p53 gene codon 72 polymorphism was a biomarker associated with esophageal squamous cell carcinoma (ESCC) and its relationship with smoking status in China. The p53 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism among 673 patients with ESCC and 694 healthy controls. The association between p53 genotypes and risk of developing ESCC was estimated by odds ratios (OR) and their 95% confidence intervals (CIs) computed by logistic regression. Compared with Arg/Arg homozygotes, Pro/Pro homozygotes had a nearly twofold increased risk (adjusted OR, 1.83; 95% CI, 1.35-2.48). For the Pro/Arg heterozygotes, there was no evident increased risk (adjusted OR, 1.01, 95% CI, 0.78-1.30). Furthermore, the risk associated with the Pro/Pro variant genotype was more pronounced in younger patients at diagnosis (= 45 years) (OR, 7.4; 95% CI, 1.44-37.89, P = 0.02), in women (OR, 3.15; 95% CI, 1.52-4.53, P = 0.02) and in non-smokers (OR, 2.49; 95% CI, 1.58-3.94) and light smokers (OR, 2.13; 95% CI, 1.15-3.93). But tests for homogeneity between smoking-related OR showed no significant differences (P = 0.4). The p53 gene codon 72 Pro/Pro genotype was significantly associated with the increased risk of ESCC in a Chinese mainland population and may be an independent factor in susceptibility to ESCC. The association was especially noteworthy in women and in younger patients.

Decomposition of soil organic carbon (SOC) is a critical component of the global carbon cycle, and accurate estimates of SOC decomposition are important for forest carbon modeling and ultimately for decision making relative to carbon sequestration and mitigation of global climate change. We determined the major pools of SOC in four sites representing major forest types in China: temperate forests at Changbai Mountain (CBM) and Qilian Mountain (QLM), and sub-tropical forests at Yujiang (YJ) and Liping (LP) counties. A 90-day laboratory incubation was conducted to measure CO(2) evolution from forest soils from each site, and data from the incubation study were fitted to a three-pool first-order model that separated mineralizable soil organic carbon into active (C(a)), slow (C(s)) and resistant (C(r)) carbon pools. Results indicate that: (1) the rate of SOC decomposition in the sub-tropical zone was faster than that in the temperature zone, (2) The C(a) pool comprised approximately 1-3% of SOC with an average mean residence time (MRT) of 219 days. The C(s) pool comprised approximately 25-65% with an average MRT of 78 yr. The C(r) pool accounted for approximately 35-80% of SOC, (3) The YJ site in the sub-tropical zone had the greatest C(a) pool and the lowest MRT, while the QLM in the temperature zone had the greatest MRT for both the C(a) and C(s) pools. The results suggest a higher capacity for long-term C sequestration as SOC in temperature forests than in sub-tropical forests.

The integrated terrestrial ecosystem C-budget model (InTEC) developed by Chen and co-workers has been used successfully to predict carbon dynamics of forests in Canada. It was tested here for forest soil organic carbon (SOC) density of China's northern temperate zone and southern subtropical zone. The results show that the simulated SOC density is highly correlated and in broad agreement with observations in Liping and in Changbaishan, representing the southern subtropical zone and the northern temperate zone in China, respectively. SOC density ranged from 2.2 to 11.2 kg/m(2) in Liping and from 3.4 to 14.8 kg/m(2) in Changbaishan. The correlation coefficients (r(2)) are 0.63 (N=16) and 0.76 (N=14) between the simulated and measured data in Liping and Changbaishan, respectively. The SOC densities under different vegetation types in Liping decrease in the order of mixed forest, broadleaf forest, Chinese fir, couch grass, and Chinese redpine, and in Changbaishan in the order of mixed forest, silver fir, larch forest, and birch forest.

This study examined HIV-related knowledge and attitudes among 524 randomly selected adult residents from 12 rural Chinese communities where HIV infection among plasma donors has been reported. Most participants were familiar with the main routes of HIV transmission but had substantial misconceptions about risk of HIV transmission through casual social contacts. Higher score of misconception and being older and married independently predicted stronger discriminatory attitude. Intervention programs with focus on eliminating misconceptions about HIV transmission may reduce stigma.

Adhesion of tumor cells to endothelial cells is known to be involved in the hematogenous metastasis of cancer, which is regulated by hypoxia. Hypoxia is able to induce a significant increase in free intracellular Ca2+ levels in both tumor cells and endothelial cells. Here, we investigate the regulatory effects of calmodulin (CaM), an intracellular calcium mediator, on tumor cell-endothelial cell adhesion under hypoxic conditions. Hypoxia facilitates HeLa cell-ECV304 endothelial cell adhesion, and results in actin cytoskeleton rearrangement in both endothelial cells and tumor cells. Suppression of CaM activation by CaM inhibitor W-7 disrupts actin cytoskeleton organization and CaM distribution in the cell-cell contact region, and thus inhibits cell-cell adhesion. CaM inhibitor also downregulates hypoxia-induced HIF-1-dependent gene expression. These results suggest that the Ca2+ -CaM signaling pathway might be involved in tumor cell-endothelial cell adhesion, and that co-localization of CaM and actin at cell-cell contact regions might be essential for this process under hypoxic stress.

It has been well established that serotonin (5-hydroxytryptamine, 5-HT) plays a key role in neuro-endocrine-immune networks, mostly through its receptors and/or transporters. Although the presence of 5-HT receptor mRNAs in peripheral blood mononuclear cells (PBMCs) of rhesus monkeys has been reported, there is little information about serotonin transporter (SERT) expression by these cells. To examine SERT expression at the transcription and translation level, one-step RT-PCR, confocal microscopy and flow cytometry were used to detect SERT mRNA and protein expression by rhesus monkey PBMCs. It was found that SERT mRNA could be detected by RT-PCR from all of the rhesus macaque PBMC RNA samples and the nucleotide sequence of the amplicons was identical to the published SERT mRNA sequence. Low level SERT immunoreactivity was also demonstrated on the surface of rhesus PBMCs by confocal microscopy. Almost all lymphocytes and most monocytes were positive for SERT by flow cytometry. In the 2 rhesus macaques examined by multicolor flow cytometry, SERT(bright) cells were more than 84%, 94%, and 96% among CD20+, CD3+, and CD3+CD4+ lymphocytes respectively. These data demonstrate expression of SERT by rhesus macaque PBMCs, and indicate that rhesus macaques would be suitable models to test the in vivo immune regulatory effects of 5-HT or drugs targeting SERT.

To define the renal tissue changes in stone-forming patients with distal renal tubular acidosis (dRTA), we performed intra-operative papillary and cortical biopsies in five patients. The main abnormalities were plugging of inner medullary collecting ducts (IMCD) and Bellini ducts (BD) with deposits of calcium phosphate in the form of apatite; epithelial cell injury and loss was marked. Plugged ducts were surrounded by interstitial fibrosis, but the fibrosis was generalized, as well, and was a main feature of the histopathology even when plugging was not present. In contrast, common idiopathic calcium oxalate stone formers (SF) never manifest intra-tubule crystals or interstitial fibrosis. Patients with brushite (calcium monohydrogen phosphate) stones and those with cystine stones have many fewer IMCD and BD plugged with apatite (or cystine, in cystinuria), and interstitial fibrosis is limited to the regions around plugged ducts. Patients with dRTA often present a radiographic picture of nephrocalcinosis. Our direct surgical observations reveal that these may be surgically removable stones, especially in patients with well preserved renal function. In all, dRTA SF have a more diffuse papillary renal disease than other SF thus studied, and are also unusual for the degree of interstitial fibrosis.

Thermal properties of food systems at high pressure (HP) are important in the design and operation of HP processing equipment. Available techniques for thermal property evaluation under HP conditions are still very limited. In this study, a dual-needle line-heat-source (DNL) device was installed in an HP vessel to evaluate thermal conductivity (k), diffusivity (alpha), and volumetric heat capacity (C(pV)) of foods at high pressure. The DNL probe was calibrated using glycerin (0.1 MPa) and 2% (w/w) agar gel (0.1 to 350 MPa) at 5 and 25 degrees C. Calibration results showed a good correlation with the reference data of pure water: R(2)= 0.966 for thermal conductivity and R(2)= 0.837 for diffusivity, and a small standard deviation of relative error (3.18%) for the volumetric heat capacity. Fresh potato and cheddar cheese were used as test samples at 5 degrees C at selected pressure levels (0.1 to 350 MPa). The potato samples gave thermal properties very close to those of pure water, but much higher than those of the cheese. The k and alpha values of both potato and cheese increased with pressure and a 2nd-order polynomial well fitted their pressure dependency. The volumetric heat capacity data did not show a clear pressure-dependency trend. The experimental system worked well for the evaluation of thermal properties at pressures up to 350 MPa.

We amplified gag sequences from 66 individuals infected with HIV-1 CRF07&#95;BC during 2003-2005 in the Xinjiang region of China. A novel deletion of 7aa (including a KELY motif) in the central region of the CRF07&#95;BC gag p6 domain was detected, which has not been reported in other HIV-1 subtypes. Further deletions of up to 13aa (including KQE and KELY motifs) was also found in this domain, representing the biggest natural deletion up to now. Moreover, the CD4+ count and viral load level indicated that 1-13aa deletions in CRF07&#95;BC gag p6 do not have a significant effect on viral replication and fitness.

Background: Despite previous studies, uncertainty has persisted about the role of thymidylate synthase (TS) and p53 status as markers of prognosis in colorectal cancer (CRC)., Patients and Methods: A total of 967 patients accrued to a large adjuvant trial in CRC were included in a prospectively planned molecular substudy, and of them, 59% had rectal cancer and about 90% received adjuvant chemotherapy (either systemically or randomly allocated to intraportal 5-fluorouracil infusion or both). TS and p53 status were determined, blinded to any clinical data, by immunohistochemistry using a validated polyclonal antibody or the DO-7 clone, respectively, and their relationships with overall survival were examined., Results: High TS expression was observed in 58% and overexpression of p53 in 60% of tumours. TS expression correlated with tumour stage, and p53 overexpression, with rectal cancers. There was no evidence that either marker was significantly associated with survival by either univariate (TS hazard ratio (HR) = 0.94, 95% CI 0.76-1.18 and P = 0.6 and p53 HR = 0.98, 95% CI 0.78-1.23 and P = 0.9) or multivariate analyses (TS HR = 0.99, 95% CI 0.79-1.25 and P = 0.9 and p53 HR = 0.98, 95% CI 0.78-1.23 and P = 0.8)., Conclusions: Neither TS nor p53 expression has significant prognostic value in the adjuvant setting of CRC.

We have biopsied the papillae of patients who have cystine stones asking if this stone type is associated with specific tissue changes. We studied seven cystine stone formers (SF) treated with percutaneous nephrolithotomy using digital video imaging of renal papillae for mapping and obtained papillary biopsies. Biopsies were analyzed by routine light and electron microscopy, infrared spectroscopy, electron diffraction, and micro-CT. Many ducts of Bellini (BD) had an enlarged ostium, and all such were plugged with cystine crystals, and had injured or absent lining cells with a surrounding interstitium that was inflamed to fibrotic. Crystal plugs often projected into the urinary space. Many inner medullary collecting ducts (IMCD) were dilated with or without crystal plugging. Apatite crystals were identified in the lumens of loops of Henle and IMCD. Abundance of interstitial Randall's plaque was equivalent in amount to that of non-SF. In the cortex, glomerular obsolescence and interstitial fibrosis exceeded normal. Cystine crystallizes in BD with the probable result of cell injury, interstitial reaction, nephron obstruction, and with the potential of inducing cortical change and loss of IMCD tubular fluid pH regulation, resulting in apatite formation. The pattern of IMCD dilation, and loss of medullary structures is most compatible with such obstruction, either from BD lumen plugs or urinary tract obstruction from stones themselves.

Several genome-wide screens have indicated the presence of an autism susceptibility locus within the distal long arm of chromosome 7 (7q). Mapping at 7q22 within this region is the candidate gene reelin (RELN). RELN encodes a signaling protein that plays a pivotal role in the migration of several neuronal cell types and in the development of neural connections. Given these neurodevelopmental functions, recent reports that RELN influences genetic risk for autism are of significant interest. The total data set consists of 218 Caucasian families collected by our group, 85 Caucasian families collected by AGRE, and 68 Caucasian families collected at Tufts University were tested for genetic association of RELN variants to autism. Markers included five single-nucleotide polymorphisms (SNPs) and a repeat in the 5'-untranslated region (5'-UTR). Tests for association in Duke and AGRE families were also performed on four additional SNPs in the genes PSMC2 and ORC5L, which flank RELN. Family-based association analyses (PDT, Geno-PDT, and FBAT) were used to test for association of single-locus markers and multilocus haplotypes with autism. The most significant association identified from this combined data set was for the 5'-UTR repeat (PDT P-value=0.002). These analyses show the potential of RELN as an important contributor to genetic risk in autism.

Apolipoprotein E (APOE) is the only universally confirmed susceptibility gene for late-onset Alzheimer disease (LOAD), although many loci are believed to modulate LOAD risk. The genetic homogeneity of isolated populations, such as the Amish, potentially provide increased power to identify LOAD susceptibility genes. Population homogeneity in these special populations may reduce the total number of susceptibility genes contributing to the complex disorder, thereby increasing the ability to identify any one susceptibility gene. Dementia in the Amish is clinically indistinguishable from LOAD in the general population. Previous studies in the Amish demonstrated a significantly decreased frequency of the APOE-4 susceptibility allele, but significant familial clustering of dementia [M.A. Pericak-Vance, C.C. Johnson, J.B. Rimmler, A.M. Saunders, L.C. Robinson, E.G. D'Hondt, C.E. Jackson, J.L. Haines, Alzheimer's disease and apolipoprotein E-4 allele in an Amish population, Ann. Neurol. 39 (1996) 700-704]. These data suggested that a genetic etiology independent of APOE may underlie the dementia observed in this population. In the present analysis, we focused on a large, multiplex, inbred Amish family (24 sampled individuals; 10 of whom are affected). We completed a genomic screen to identify novel LOAD loci (n=316 genetic markers), using both model-dependent "affecteds-only" analysis (dominant and recessive) and model-independent affected relative pair analysis. Interesting results (lod>1.5 or p<0.01) were obtained for markers on eight chromosomes (2q, 5q, 6q, 7p, 8p, 8q, 11p, 18p, 18q, and 19q). The highest overall score was a multipoint lod score of 3.1 on chromosome 11p. Most regions we identified were not previously detected by genomic screens of outbred populations and may represent population-specific susceptibilities to LOAD. These loci are currently under further investigation in a study of LOAD including additional Amish families.

This study examines the basis of resistance and sensitivity of normal and transformed cells to histone deacetylase inhibitor (HDACi)-induced cell death, specifically the role of caspases and thioredoxin (Trx). An important attribute of HDACis is that they induce cancer cell death at concentrations to which normal cells are relatively resistant, making them well suited for cancer therapy. The mechanism underlying this selectivity has not been understood. In this study we found that the HDACi suberoylanilide hydroxamic acid (SAHA) and MS-275, a benzamide, cause an accumulation of reactive oxygen species (ROS) and caspase activation in transformed but not normal cells. Inhibition of caspases does not block HDACi-induced cell death. These studies provide a possible mechanism that can explain why normal but not certain transformed cells are resistant to HDACi-induced cell death. The HDACi causes an increase in the level of Trx, a major reducing protein for many targets, in normal cells but not in transformed cells. The SAHA-induced increase in Trx activity in normal cells is associated with no increase in ROS accumulation. Transfection of transformed cells with Trx small interfering RNA caused a marked decrease in the level of Trx protein with an increase in ROS, a decrease in cell proliferation, and an increase in sensitivity to SAHA-induced cell death. Thus, Trx, independent of the caspase apoptotic pathway, is an important determinant of resistance of cells to HDACi-induced cell death.

Activating mutations in the RAS oncogenes are among the most common genetic alterations in human cancers, including patients with acute lymphoblastic leukemia (ALL). We sought to define the frequency and spectrum, and possible prognostic importance, of N- and K-RAS mutations in children with ALL treated with contemporary therapy. Leukemic blast DNA from 870 children was analyzed for the presence of activating mutations in the N- or K-RAS oncogenes using a sensitive mutation detection algorithm. RAS mutations were present in the blasts of 131 (15.1%) pediatric ALL patients. The spectrum of mutations included 81 (9.3%) mutations of codons 12/13 of N-RAS, 12 (1.4%) mutations of codon 61 of N-RAS, 39 (4.5%) mutations of codons 12/13 of K-RAS, and 2 (0.2%) mutations of codon 61 of K-RAS. The presence of N- or K-RAS mutations was not associated with white blood cell count at diagnosis, sex, race, extramedullary testicular involvement, central nervous system disease, or NCI/CTEP ALL Risk Group. Patients with an exon 1 K-RAS mutation (codons 12/13) were significantly younger at diagnosis (P=0.001) and less frequently B-lineage phenotype (P=0.01). RAS mutation status did not predict overall survival, event-free survival and disease-free survival. While N- and K-RAS mutations can be identified in 15% of children with newly diagnosed ALL, they do not represent a significant risk factor for outcome using contemporary chemotherapy regimens.

Autism is a neurodevelopmental disorder characterized by stereotypic and repetitive behavior and interests, together with social and communicative deficiencies. The results of several genomic screens suggest the presence of an autism susceptibility locus on chromosome 19p13.2-q13.4. The apolipoprotein E (APOE) gene on chromosome 19 encodes for a protein, apoE, whose different isoforms (E2, E3, E4) influence neuronal growth. APOE participates in lipid transport and metabolism, repair, growth, and maintenance of axons and myelin during neuronal development. The APOE protein competes with the Reelin protein for VLDL/APOER2 receptor binding. Several studies have reported evidence for an association between autism and the Reelin gene. Based on these data we tested for association between APOE and autism using family-based association methods in a data set of 322 autism families. Three promoter, one intronic, and one 3' UTR single nucleotide polymorphisms (SNPs) in the APOE gene (-491a/t, -427c/t, -219g/t, 113c/g, and 5361c/t) as well as the APOE functional polymorphism (E2, E3, E4) were examined and failed to reveal significant evidence that autism is associated with APOE., (Copyright 2003 Wiley-Liss, Inc.)

The metastable O2-LiCoO(2) phase undergoes several reversible phase transitions upon lithium deintercalation. The first transition leads to an unusual oxygen stacking in such layered compounds. This stacking is found to be stable for 0.52 < x < or = 0.72 in Li(x)()CoO(2) and is called T(#)2. We studied this phase from a structural viewpoint using X-ray and neutron diffraction (ab initio method). The new stacking derives from the O2 one by gliding every second CoO(2) slab by (1/3, 1/6, 0). The lithium ions are found to occupy very distorted tetrahedral sites in this structure. We also discuss the possibility of this T(#)2 phase to exhibit stacking faults, whose amount depends on the method used to prepare this deintercalated phase.

Voltage-sensitive calcium channels (VSCC) open in response to external stimuli, including calcitropic hormones, that alter plasma membrane calcium (Ca2+) permeability. Ca2+ that enters the cell through these channels serves a second messenger function, eliciting cellular responses that include secretion and changes in gene expression. In osteoblasts, VSCCs serve as key regulators of Ca2+ permeability and are a major class of calcitropic hormone-sensitive Ca2+ channels present in the plasma membrane. The members of the VSCC family exist as a complex of polypeptide subunits that are comprised of a pore-forming alpha1 subunit, an intracellular beta subunit, a dimer of disulfide-linked alpha2 and delta subunits, and in some tissues, a gamma subunit. Previous studies in our laboratory have shown that the major functional alpha1 subunit present in osteoblasts is the alpha1C (CaV1.2). To determine the complement of auxiliary subunits present in rodent osteoblastic cells, we employed RT-PCR using a battery of subunit specific primers and appropriate tissue controls. Immunohistochemistry also was performed, using available subunit specific antibodies, to measure protein expression and localization. Cell types examined included MC3T3-E1 at various stages of differentiation, ROS 17/2.8 osteosarcoma, and primary cultures of rat calvarial osteoblasts. The results indicate that all cells expressed multiple beta subunit classes and alpha2delta dimers, but no gamma subunits, regardless of differentiation state. We propose a structure for the functional osteoblast VSCC that consists of alpha1, beta, alpha2delta subunits and is devoid of a gamma subunit.

Objective: To evaluate the predictive effects of the new lymph node staging system based on the ratio of the number of metastatic lymph nodes to the total number of dissected lymph nodes comparing with N-staging of the 5th TNM (UICC, 1997)., Methods: Lymph nodes harvested from operative specimens in 78 patients with gastric cancer were examined histologically. pN staging was carried out according to the two staging systems respectively. In our system, pN0, pN1, pN2, pN3 were defined as lymph node ratio(LNR) 0, <10%, <25% and >25%, respectively. All the patients were followed up and the data were analyzed statistically., Results: A total of 5388 lymph nodes were found with a mean of 69 per case (range 30-157). Lymph node metastases were detected in 59 patients (75.64%). The 3-year survival rates of pN0, pN1, pN2, pN3 of the new staging system were 100%, 68.42%, 7.58% and 6.78%, respectively., Conclusion: The lymph node ratio is a relative variable, which reflects the degree of metastasis of lymph node, and is more predictable than the UICC 1997 5th N-staging system defined as the numbers of metastatic lymph node.

A novel method for fabrication of nanometer-sized electrodes and tips suitable for scanning electrochemical microscopy (SECM) is reported. A fine etched Pt wire is coated with polyimide, which was produced by polymerization on the Pt surface initiated by heat. This method can prepare electrodes with effective radii varying from a few to hundreds of nanometers. Scanning electron microscopy, cyclic voltammetry, and SECM were used to characterize these electrodes. Well-defined steady-state voltammograms could be obtained in aqueous or in 1,2-dichloroethane solutions. This method produced the nanoelectrodes with exposed Pt on the apex, and they can also be employed as the nanotips for SECM investigations. Different sizes of Pt nanotips made by this method were employed to evaluate the kinetics of the redox reaction of Ru(NH3)6(3+) on the surface of a large Pt electrode by SECM, and the standard rate constant kappa0 of this system was calculated from the best fit of the SECM approach curve. This result is similar to the values obtained by analysis of the obtained voltammetric data.

Tabtoxin-resistance protein (TTR), an acetyltransferase from Pseudomonas syringae pv. tabaci, was overexpressed in Eschericha coli M15 and the TTR fusion protein complexed with acetyl-coenzyme A (AcCoA) was purified and crystallized. Diffraction data were collected to 3.0 A resolution in-house and the crystal was found to belong to space group P2(1), with unit-cell parameters a = 47.6, b = 66.6, c = 53.5 A, beta = 104.3 degrees. Furthermore, a selenomethionine (SeMet) TTR fusion protein derivative was overexpressed in the same expression system and its complex with AcCoA was purified in a reductive environment. The SeMet TTR derivative crystallized in two forms: the first was identical to that observed for native crystals and the second belonged to space group C2, with unit-cell parameters a = 101.7, b = 45.6, c = 84.2 A, beta = 105.8 degrees. Data from the P2(1) crystal form were collected in-house to 2.3 A resolution. Subsequently, three different wavelength data sets of the C2 crystal form to 1.55 A resolution were collected at the Advanced Photon Source at Argonne National Laboratory.

In a 0.125 mol/L phosphate (pH 6.6)/2.5 x 10(-4) mol/L 2-iodoacetamide solution, lomefloxacin yields a response of a polarographic catalytic current. The second-order derivative peak current of the catalytic wave of lomefloxacin is proportional to its concentration in the range of 1.0 x 10(-8)-1.0 x 10(-6) mol/L (r = 0.998). The sensitivity of the catalytic wave is 25-times higher than that of the corresponding reduction wave for 5.0 x 10(-7) mol/L lomefloxacin. The proposed method was applied to the determination of lomefloxacin in pharmaceutical preparations. The polarographic reduction wave is ascribed to a one-electron reduction of the C=C bond of lomefloxacin zwitterion accompanied by an acid-base equilibrium. The catalytic wave should be caused by regeneration of the lomefloxacin molecule at electrode surface due to the one-electron reduction product being further oxidized by electroreductive intermediate products of 2-iodoacetamide.

Objective: To evaluate the effect of high dose chemotherapy with or without total body irradiation (TBI) supported by autologous hematopoietic stem cell transplantation (AHSCT) for malignant lymphoma., Methods: From June 1991 to April 2000, 32 malignant lymphoma (ML) patients were treated with AHSCT. Their ages ranged from 19 to 52 years (median 37). There were 23 non-Hodgkin's lymphoma (NHL) and 9 Hodgkin's lymphom (HD). Twelve patients received autologous bone marrow transplantation (ABMT) and 20 patients received autologous peripheral blood stem cell transplantation (APBSCT). In the latter, the mobilization plan was carboplatin and etoposide (CE) or CHOP (CTX, ADM, VCR, PDN) plus G/GM-CSF 10 micrograms/kg/d in high dose chemotherapy. BEAM (BCNU, Vp-16, AraC, MEL) or MEL 140 mg/m2 + Vp-16 200 mg + total body irradiation (TBI) 8 Gy., Results: Up to May 2000, hematopoietic reconstitution was reestabished in 100% (32/32) patients after AHSCT, with median follow-up of 1,020 days (40-3,240). The 1- and 2-year disease-free survivals (DFS) were 78.1%(25/32) and 46.9% (15/32) with the longest survival of 8 years. Even though eight patients have developed relapse, there is no transplantation-related death., Conclusion: High dose chemotherapy and radiotherapy supported by autologous hematopoietic stem cell transplantation (AHSCT) for high risk, relapsed or sensitive ML, being highly efficient, should be the first indication. It is better than the standard conventional treatment. The hematopoietic reconstitution of autologous peripheral blood stem cell transplantation (APBSCT) is quicker than autologous bone marrow transplantation (ABMT). No significant difference between chemotherapy alone and chemotherapy plus total body irradiation is observed.

Humans express three distinct collagenases, MMP-1, MMP-8, and MMP-13, that initiate degradation of fibrillar type I collagen. We have previously reported that ultraviolet irradiation causes increased expression of MMP-1, but not MMP-13, in keratinocytes and fibroblasts in human skin in vivo. We report here that ultraviolet irradiation increases expression of MMP-8 in human skin in vivo. Western analysis revealed that levels of the full-length, 85 kDa proenzyme form of MMP-8 increased significantly within 8 h post ultraviolet irradiation (2 minimal erythema doses). Increased full-length MMP-8 protein was associated with infiltration into the skin of neutrophils, which are the major cell type that expresses MMP-8. Immunofluorescence revealed coexpression of MMP-8 and neutrophil elastase, a marker for neutrophils. Immunohistology demonstrated MMP-8 expression in neutrophils in the papillary dermis between 4 and 8 h post ultraviolet irradiation, and in the epidermis at 24 h post radiation. MMP-8 mRNA expression was not detected in nonirradiated or ultraviolet-irradiated human skin, indicating that increased MMP-8 following ultraviolet irradiation resulted from preexisting MMP-8 protein in infiltrating neutrophils. Pretreatment of skin with the glucocorticoid clobetasol, but not all-trans retinoic acid, significantly blocked ultraviolet-induced increases in MMP-8 protein levels, and neutrophil infiltration. In contrast, all-trans retinoic acid and clobetasol were equally effective in blocking ultraviolet induction of MMP-1 and degradation of collagen in human skin in vivo. Taken together, these data demonstrate that ultraviolet irradiation increases MMP-8 protein, which exists predominantly in a latent form within neutrophils, in human skin in vivo. Although ultraviolet irradiation induces both MMP-1 and MMP-8, ultraviolet-induced collagen degradation is initiated primarily by MMP-1, with little, if any, contribution by MMP-8.

Triggering of the T cell antigen receptor (TCR).CD3 complex induces its ubiquitination. However, the molecular events that lead to ubiquitin conjugation to these cell surface molecules have not been defined. Here we report that Cbl, a RING-type E3 ubiquitin-protein ligase, promotes ubiquitination of TCR zeta chain, which requires its functional variant Src homology 2 domain and an intact RING finger. The tyrosine kinase Zap-70, which binds to both TCR zeta and Cbl, plays an adaptor role in these events. Mutations in TCR zeta, Zap-70, or Cbl that disrupt the interaction between TCR zeta and Zap-70 or between Zap-70 and Cbl reduce ubiquitination of TCR zeta. Our results suggest a novel mechanism by which Cbl negatively regulates T cell development and activation by inducing ubiquitination of the TCR.CD3 components.

Synthetic chemosensors hold great potential in many diagnostic applications. In this study, we describe the design and preparation of the first encoded combinatorial library of chemosensors for tripeptides. Subsequent screening of the library resulted in the discovery of novel chemosensors able to distinguish between random tripeptides.

We have generated anti-HER2 (ErbB2) immunoliposomes (ILs), consisting of long circulating liposomes linked to anti-HER2 monoclonal antibody (MAb) fragments, to provide targeted drug delivery to HER2-overexpressing cells. Immunoliposomes were constructed using a modular strategy in which components were optimized for internalization and intracellular drug delivery. Parameters included choice of antibody construct, antibody density, antibody conjugation procedure, and choice of liposome construct. Anti-HER2 immunoliposomes bound efficiently to and internalized in HER2-overexpressing cells in vitro as determined by fluorescence microscopy, electron microscopy, and quantitative analysis of fluorescent probe delivery. Delivery via ILs in HER2-overexpressing cells yielded drug uptake that was up to 700-fold greater than with non-targeted sterically stabilized liposomes. In vivo, anti-HER2 ILs showed extremely long circulation as stable constructs in normal adult rats after a single i.v. dose, with pharmacokinetics that were indistinguishable from sterically stabilized liposomes. Repeat administrations revealed no increase in clearance, further confirming that ILs retain the long circulation and non-immunogenicity of sterically stabilized liposomes. In five different HER2-overexpressing xenograft models, anti-HER2 ILs loaded with doxorubicin (dox) showed potent anticancer activity, including tumor inhibition, regressions, and cures (pathologic complete responses). ILs were significantly superior vs. all other treatment conditions tested: free dox, liposomal dox, free MAb (trastuzumab), and combinations of dox+MAb or liposomal dox+MAb. For example, ILs produced significantly superior antitumor effects vs. non-targeted liposomes (P values from <0.0001 to 0.04 in eight separate experiments). In a non-HER2-overexpressing xenograft model (MCF7), ILs and non-targeted liposomal dox produced equivalent antitumor effects. Detailed studies of tumor localization indicated a novel mechanism of drug delivery for anti-HER2 ILs. Immunotargeting did not increase tumor tissue levels of ILs vs. liposomes, as both achieved very high tumor localization (7.0-8.5% of injected dose/g tissue) in xenograft tumors. However, histologic studies using colloidal-gold labeled ILs demonstrated efficient intracellular delivery in tumor cells, while non-targeted liposomes accumulated within stroma, either extracellularly or within macrophages. In the MCF7 xenograft model lacking HER2-overexpression, no difference in tumor cell uptake was seen, with both ILs and non-targeted liposomes accumulating within stroma. Thus, anti-HER2 ILs, but not non-targeted liposomes, achieve intracellular drug delivery via receptor-mediated endocytosis, and this mechanism is associated with superior antitumor activity. Based on these results, anti-HER2 immunoliposomes have been developed toward clinical trials. Reengineering of construct design for clinical use has been achieved, including: new anti-HER2 scFv F5 generated by screening of a phage antibody library for internalizing anti-HER2 phage antibodies; modifications of the scFv expression construct to support large scale production and clinical use; and development of methods for large-scale conjugation of antibody fragments with liposomes. We developed a scalable two-step protocol for linkage of scFv to preformed and drug-loaded liposomes. Our final, optimized anti-HER2 ILs-dox construct consists of F5 conjugated to derivatized PEG-PE linker and incorporated into commercially available liposomal doxorubicin (Doxil). Finally, further studies of the mechanism of action of anti-HER2 ILs-dox suggest that this strategy may provide optimal delivery of anthracycline-based chemotherapy to HER2-overexpressing cancer cells in the clinic, while circumventing the cardiotoxicity associated with trastuzumab+anthracycline. We conclude that anti-HER2 immunoliposomes represent a promising technology for tumor-targeted drug delivery, and that this strategy may also be applicable to other receptor targets and/or using other delivered agents.

Objective: To investigate mammanglobin (hMAM) mRNA as a marker for the detection of carcinoma cells by reverse transcriptase chain reaction (RT-PCR) in the peripheral blood of breast cancer patients., Methods: Blood samples from 63 breast cancer patients obtained at various stages of their disease, blood samples from 8 breast hyperplasia, 5 breast adenofibroma, 25 other cancers (stomach, colon, esophagus, lung, ovary) and 31 healthy volunteers were screened for hMAM mRNA by a nested RT-PCR combined with fluorescence quantitative PCR (FQ-PCR) assay., Results: Among 63 breast carcinoma patients, 19(30.2%) were RT-PCR positive for hMAM mRNA which was elevated with the tumor stage. None of the other cancer patients or those suffering from benign breast diseases were positive but only 1 of the 31 healthy volunteers gave detectable hMAM mRNA findings. hMAM mRNA was not detectable in the peripheral blood after operation in 6(32%) of 19 patients whose pre-operative test had been positive even though the hMAM mRNA expression increased with tumor stage. These results did not correlate with patients' stage, estrogen or pregnant receptor status., Conclusion: hMAM transcripts are detectable in the peripheral blood of some breast cancer patients. hMAM may be a novel candidate and a clinically useful breast tumor marker, especially in detecting micrometastasis.

Objective: To investigate the therapeutic effects of Adenovirus-transferred antisense c-myc (Ad-Asmyc) on hepatocellular cancer cells and its mechanism., Methods: Human hepatocellular cancer cell line BEL-7402 and QSG-7701 were transfected with Ad-Asmyc. Cell growth curve and coloning formation were used to investigate the therapeutic effects of Ad-Asmyc in Vitro. The treating effects of Ad-Asmyc on transplanted tumor in nude mice was also studied. RT-PCR and Western blot were used to detect relating gene expression. Flowcytometry, DNA ladder and tunnel assay were used to study the cell cycle and apoptosis., Results: The expression of c-myc in cancer cells was reduced after infected Ad-Asmyc. Ad-Asmyc had obviously inhibiting effects on the growth of two hepatocellular carcinoma cell lines in vitro and also in vivo. It can induced cell cycle arrest (G1 arrest) and apoptosis., Conclusion: Ad-Asmyc can inhibit HCC cell proliferation and induce apoptosis, so has obvious therapeutic effects on hepatocellular cancer cells.

We have previously identified five Na, K-ATPase beta1-mRNA species that are expressed in the rat heart, kidney, and brain. These mRNAs which are unequal in their abundance have an identical coding region but differ in the length of their 5'- and 3'-untranslated regions (UTRs). In this study we examined the possibility that the beta1-mRNA species exhibit differential translational efficiencies. We constructed expression plasmids encoding each of the five mRNAs and transcribed and translated them in vitro. Using rabbit reticulocyte system we determined the translation of the different mRNAs under conditions optimized for each beta1-cRNA and under an equivalent (standard) condition. The longest beta1-cRNA species (initiating at the first transcription start site and ending at the last [fifth] poly(A) site) exhibited the lowest relative translational efficiency averaging 0.2 +/- 0.05 units/mol of cRNA compared to the shortest beta1-cRNA species initiating at the first transcription start site and ending at the first poly(A) signal (with an assigned relative value of 1.0). These results suggested that the different translation rates of beta1-mRNAs may be due to their 3'-UTRs. To further define the role of beta1-3'-UTR, chimeric luciferase constructs containing different segments of the beta1-3'-UTR were transiently transfected into Clone 9 cells. Compared to the chimeric construct containing the shortest beta1-3'-UTR segment (ending at the first poly(A) site), the construct containing the full-length beta1-3'-UTR exhibited a luciferase expression of 0.23 +/- 0.04. To control for potential changes in the abundance of the expressed chimeric mRNAs which may lead to differences in luciferase expression, luciferase activity was normalized against chimeric luciferase-mRNA content measured in mixtures of cells stably transfected with the above constructs. The ratio of luciferase activity/chimeric luciferase-mRNA content in cells expressing the construct containing the entire beta1-3'-UTR region was 0.17 that in cells expressing chimeric luciferase mRNA containing beta1-3'-UTR up to the first poly(A) signal (P < 0.05). We conclude that the translational efficiency of the different beta1-mRNA species is negatively regulated by the 3'-UTR of the mRNA and that a regulating region appears to be localized between the second and fifth poly(A) signals of beta1-mRNA., (Copyright 2001 Academic Press.)

Growth factors interact with their cell surface receptors and activate the enzyme PI 3-kinase (PI 3-K) resulting in the formation of 3-phosphorylated phosphatidylinositols, which in turn activate the serine/threonine kinase AKT/PKB. AKT functions, in part, to promote cell survival by phosphorylating the BCL-2 family member BAD and the cell death pathway enzyme, caspase-9. Although induction of apoptosis by ultraviolet (UV) irradiation is well documented, little is known about UV activation of cell survival pathways in human skin cells. We have investigated whether UV activates the PI 3-K/AKT pathway in human skin in vivo. UV irradiation (2MED from UVB source) stimulated PI 3-kinase activity within 15 min. PI 3-K activity was maximal (2.5-fold, n=6) 30 min post UV and remained elevated for 4 h. UV stimulated AKT activity within 30 min. Maximal activity (4-fold, n=11) was observed 1 h post UV. UV also stimulated phosphorylation of the downstream AKT effectors, S6 kinase and BAD. S6 kinase was maximally stimulated 4 h post UV (15-fold, n=6). Increased BAD phosphorylation was observed 1 h post UV and remained elevated for 4 h. Western blot analysis revealed that UV-induced phosphorylation of BAD at Ser112, a site known to be phosphorylated by AKT. Inhibitors of EGFR and PI 3-kinase blocked UV-induced phosphorylation of BAD, suggesting that EGFR mediates UV-activated cell survival pathway. Collectively, both positive and negative roles for UV activation of the PI 3-K/AKT pathway in human skin can be envisioned. The PI 3-K/AKT pathway likely plays a critical role in balancing UV-induced apoptotic signals, thereby preventing widespread skin cell death. Conversely UV activation of the PI 3-K/AKT pathway may enhance survival of mutated cells, thereby promoting skin cancer, as has been found in several other types of cancer.

Type I and type III procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced procollagen synthesis. In the present study, we investigated type I procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I procollagen gene expression by in situ hybridization and for type I procollagen protein by immunostaining. Both mRNA and protein were reduced ( approximately 65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I procollagen production was because of inherently reduced capacity of skin fibroblasts in severely photodamaged forearm skin to synthesize procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I procollagen synthesis. For these studies, fibroblasts from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of fibroblasts were isolated from the two skin sites. Fibroblasts from the two sites had similar growth capacities and produced virtually identical amounts of type I procollagen protein. These findings indicate that the lack of type I procollagen synthesis in sun-damaged skin is not because of irreversible damage to fibroblast collagen-synthetic capacity. It follows, therefore, that factors within the severely photodamaged skin may act in some manner to inhibit procollagen production by cells that are inherently capable of doing so. Interactions between fibroblasts and the collagenous extracellular matrix regulate type I procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. Fibroblasts are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is approximately 3.6-fold greater in photodamaged skin than in sun-protected skin, and some fibroblasts are surrounded by debris. To model this situation, skin fibroblasts were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal fibroblasts, whereas intact collagen did not. Fibroblasts cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (22 and 17% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) fibroblasts from photoaged and sun-protected skin are similar in their capacities for growth and type I procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I procollagen synthesis.

The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis, some of which included fatalities caused by haemolytic uraemic syndrome. Close to 75,000 cases of O157:H7 infection are now estimated to occur annually in the United States. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7 (ref. 4). Here we have sequenced the genome of E. coli O157:H7 to identify candidate genes responsible for pathogenesis, to develop better methods of strain detection and to advance our understanding of the evolution of E. coli, through comparison with the genome of the non-pathogenic laboratory strain E. coli K-12 (ref. 5). We find that lateral gene transfer is far more extensive than previously anticipated. In fact, 1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions--all of which could be targets for surveillance.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, acting via the GABAA receptors. The GABAA receptors are comprised of several different homologous subunits, forming a group of receptors that are both structurally and functionally diverse. Three of the GABAA receptor subunit genes (GABRB3, GABRA5 and GABRG3) form a cluster on chromosome 15q11-q13, in a region that has been genetically associated with autistic disorder (AutD). Based on these data, we examined 16 single nucleotide polymorphisms (SNPs) located within GABRB3, GABRA5 and GABRG3 for linkage disequilibrium (LD) in 226 AutD families (AutD patients and parents). Genotyping was performed using either OLA (oligonucleotide ligation assay), or SSCP (single strand conformation polymorphism) followed by DNA sequencing. We tested for LD using the Pedigree Disequilibrium Test (PDT). PDT results gave significant evidence that AutD is associated with two SNPs located within the GABRG3 gene (exon5&#95;539T/C, p=0.02 and intron5&#95;687T/C, p=0.03), suggesting that the GABRG3 gene or a gene nearby contributes to genetic risk in AutD.

Interleukin-18 (IL-18) and IL-12 have been shown to play an important role in the induction of interferon-gamma (IFN-gamma). IFN-gamma induces the proliferation of T cells and natural killer (NK) cells and augments the Th1 immune cascade. The role of IL-18 and IL-12 in the induction of IFN-gamma following allogeneic heart transplantation has not been described. We sought to characterize the IL-12 and IL-18 response to murine allogeneic heart transplantation, particularly with respect to IFN-gamma production and histologic transplant rejection. Forty-eight heterotopic heart transplants were performed in two groups of mice: syngeneic C3H/HeN to C3H/HeN mice and allogeneic BALB/C to C3H/HeN mice. Transplants were followed out to 2, 6, 10, and 14 days. Six transplants were performed in each group. Serum and splenic samples were used to evaluate the cytokine response by ELISA. Explanted heart tissue was processed for evidence of histologic rejection, and RT-PCR was performed to evaluate the IL-12, IL-18, and IFN-gamma signal qualitatively. Analysis of variance (ANOVA), Fisher's projected least significant difference (PLSD) was used for statistical analysis. Transplant rejection occurred in the allogeneic group histologically by day 6 and clinically by day 10. Serum IFN-gamma levels rose significantly by day 6 in the allogeneic group and then continued to rise in the splenocyte cultures. Serum IL-18 also rose significantly in the allogeneic group at day 6 compared with syngeneic group. RT-PCR revealed that the allogeneic tissue contained an increased signal for IL-12, IL-18, and IFN-gamma beginning at day 6 and peaking at day 10 after transplant. Beginning 6 days after transplantation, IL-12 and IL-18 appear to play a significant role in the induction of IFN-gamma in allogeneic heart transplants.

A multiple analytic approach may be useful for analyzing complex traits since different methods extract both similar and distinct, but complementary pieces of information from genome screen data on extended pedigrees. We examined the usefulness of combining p-values both across methods and across adjacent markers, taking into account the observed correlation structure among these p-values. To this end, we employed the recently proposed truncated product method [Zaykin et al., Genet Epidemiol, in press]. It appears that this approach is helpful for visualizing priority regions for follow-up analysis and reducing the number of false-positive linkage signals.

The a-factor of Saccharomyces cerevisiae is a dodecapeptide pheromone (YIIKGVFWDPAC(Farnesyl)-OCH(3), 1), in which post-translational modification with a farnesyl isoprenoid and carboxymethyl group is required for full biological activity. This peptide has been used as a model system to explore the biological function of the farnesylcysteine moiety, which is found on and required for the biological activity of many key mammalian proteins. The objective of this particular study was the determination of the biological effect of double bond isomerization of the natural E, E-farnesyl moiety on the biological activity of the a-factor. A unified, stereoselective synthetic route to the three geometric isomers of E,E-farnesol (12, 13, and 14) has been developed. The key feature of this synthesis is the ability to control the stereochemistry of triflation of the beta-ketoester 22 to give either 23 or 25. The three farnesol isomers were converted to the corresponding isomeric a-factors (9, 10 and 11) via a modified version of a previously utilized synthetic route. Biological evaluation of these peptides indicates that, surprisingly, all three possess nearly equivalent activity to the natural a-factor bearing the E,E-farnesyl moiety.

New outbreaks of human immunodeficiency virus type 1 (HIV-1) among injecting drug users (IDUs) are spreading in China along heroin trafficking routes. Recently, two separate HIV-1 epidemics among IDUs were reported in Guangxi, Southern China, where partial sequencing of the env gene showed subtype C and circulating recombinant form (CRF) AE. We evaluated five virtually full-length HIV-1 genome sequences from IDUs in Guangxi to determine the genetic diversity and the presence of intersubtype recombinants. Sequence analysis showed two geographically separated, highly homogeneous HIV-1 strains. B/C intersubtype recombinants were found in three IDUs from Baise City, in a mountainous region near the Yunnan-Guangxi border. These were mostly subtype C, with portions of the capsid and reverse transcriptase (RT) genes from subtype B. The subtype B portion of the capsid was located in the N-terminal domain, which has been shown to influence virus core maturation, virus infectivity, and binding to cyclophilin A, whereas the subtype B portion of RT was located in the palm subdomain, which is the active site of the enzyme. These BC recombinants differed from a BC recombinant found in Xinjiang Province in northwestern China. CRF AE strains were found in IDUs from Nanning, the capital of Guangxi, and in IDUs from Pingxiang City near the China-Vietnam border. The AE and BC recombinants were both remarkable for their low interpatient diversity, less than 1% for the full genome. Rapid spread of HIV-1 among IDUs may foster the emergence of highly homogeneous strains, including novel recombinants in regions with multiple subtypes.

Objective: To investigate the prevalent status of HIV-1 subtype E epidemic among different populations in Shenzhen, China., Methods: HIV-1 proviral DNA from peripheral blood mononuclear cells (PBMCs) of 3 HIV-infected individuals found in Shenzhen was amplified by nested-PCR. The C2-V3 and its flanking region (350-450 nucleotides) of HIV- 1 env gene were sequenced directly from the PCR products and analysed., Results: These 3 cases were confirmed to be infected with HIV-1 subtype E strain (sz-E). The innergroups distance of sz-E was 2.6%. In comparison with the consensus sequence of international A-E subgroups and some of subtype B epidemic in China, the genetic distances among sz-E and each of A-D subgroups and some of subtype B were more than 24 %. Furthermore, the phylogenetic tree analysis showed that sz-E clustered closely to Econ., Conclusions: These findings suggested that the epidemic time for HIV-1 subtype E, which was mainly prevalent among IDUs population and some professional blood donors, had been about 2-3 years in Shenzhen.

A molecular epidemiology study was conducted among more than 100 human immunodeficiency virus type 1 (HIV-1) subtype C seropositive intravenous drug users (IDUs) from China. Genotyping based on the envelope C2V3 coding region revealed the highest homology of the most prevalent virus strains circulating throughout China to subtype C sequences of Indian origin. Based on these results, a virtually full-length genome representing the most prevalent class of clade C strains circulating throughout China was directly amplified from peripheral blood mononuclear cells of a selected HIV-infected IDU and subcloned. Sequence analysis identified a mosaic structure, suggesting extensive intersubtype recombination events between genomes of the prevalent clade C and (B')-subtype Thai virus strains of that geographic region. Recombinant Identification Program analysis and phylogenetic bootstrapping suggested that there were 10 breakpoints (i) in the gag-pol coding region, (ii) in vpr and at the 3' end of the vpu gene, and (iii) in the nef open reading frame. (B')-sequences therefore include (i) several insertions in the gag-pol coding region; (ii) 3'-vpr, the complete vpu gene, and the first exons of tat and rev; and (iii) the 5' half of the nef gene. Breakpoints located in the vpr/vpu coding region as well as in the nef gene of 97cn54 were found at almost identical positions of all subtype C strains isolated from IDUs living in different areas of China, suggesting a common ancestor for the C/B' recombinant strains. More than 50% of well-defined subtype B-derived cytotoxic T-lymphocyte epitopes within Gag and Pol and 10% of the known epitopes in Env were found to exactly match sequences within in this clade C/B' chimeric reference strain. These results may substantially facilitate a biological comparison of clade C-derived reference strains as well as the generation of useful reagents supporting vaccine-related efforts in China.

The expression of GADD45 was examined in cultured skin keratinocytes and in human skin in vivo following UV irradiation. Northern blot analysis revealed that UV-induced the expression of GADD45 (alpha, beta, gamma) in a time- and dose-dependent manner. Messenger RNA of GADD45 (alpha, beta, gamma) increased within 30 min, peaked at 4 h and remained elevated for at least 8 h following UV irradiation in vitro and in vivo. Maximal induction of GADD45alpha was approximately 5-fold compared to the level in sham-irradiated controls. Similarly H2O2 and IL-1 also induced GADD45alpha expression in cultured human keratinocytes. The kinetics of induction of GADD45alpha by H2O2, IL-1beta and UV were very similar. Interestingly, UV-induced GADD45alpha expression was inhibited by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, and antioxidant, N-acetyl-L-cysteine (NAC), indicating the involvement of reactive oxygen species in UV signaling. Previously we have shown that EGF receptor activation by UV is prerequisite for subsequent activation of NADPH oxidase and generation of reactive oxygen species. We therefore examined the effect of EGF receptor inhibitor on UV-induced GADD45alpha expression. Our results showed that PD168393, a potent EGF receptor inhibitor, blocked UV-induced GADD45alpha expression. Collectively, our data suggest that UV-induced GADD45alpha expression occur via an EGF receptor-mediated oxidative pathway sensitive to antioxidant regulation.