The expression of HPV-16 E5 protein in squamous neoplastic changes in the uterine cervix

To investigate the expression of human papillomavirus type 16 (HPV-16) E5 protein in squamous neoplastic changes in the uterine cervix, the specific E5 antibody was generated and used to identify the expression of E5 protein in 40 cases of HPV-16-positive tissues and 5 previously identified HPV-negative normal cervical tissues. The results revealed that E5 protein was primarily expressed in the lower third of the epithelium in low-grade squamous intraepithelial lesions (SILs) and throughout the whole epithelium in high-grade SILs. In invasive squamous carcinoma, 60% of HPV-16-infected cancers which contained the episomal viral genome had the E5 gene, and could express E5 protein which was located throughout the whole epithelium. Previously, we documented the expression of type I growth factor receptors [ERBB1/EGFR (epidermal growth factor receptor), ERBB2, ERBB3 and ERBB4] in the full range of cervical neoplasias by immunohistochemistry assay. Hence, in this study, we extensively analyzed the correlation between the expression of E5 protein and the expression of type I growth factor receptors. Among 40 HPV-16- infected cervical neoplasias, we found that the expression of E5 protein was significantly correlated with either the expression of the ERBB1 or the ERBB4 receptor.     

Recombinant adenovirus encoding H-ras ribozyme induces apoptosis in laryngeal cancer cells through caspase- and mitochondria-dependent pathways

Previously, we designed a ribozyme that targets the H-ras oncogene at the 12th codon mutation site (Chang et al., 1997). Ribozymes have antisense molecule and site-specific ribonuclease potential. In this study, an adenoviral vector was used to transduce the H-ras ribozyme into laryngeal cancer cells (HEp-2). This served to downregulate the H-ras gene expression in which this ribozyme performed antisense activity due to HEp-2 cells containing wild-type alleles in the 12th H-ras codon. Together, our data demonstrated that the recombinant adenovirus encoding H-ras ribozyme can be broadly regarded as a cytotoxic gene therapy in laryngeal cancer cells regardless of containing wild-type or mutant ras gene. In addition, the mechanism through which the H-ras ribozyme inhibited tumor growth was apoptosis and involved both caspase- and mitochondria-mediated pathways. The activators caspase-8 and -9 as well as the effector caspase-3 in the induction phase of apoptosis and the substrate PARP of caspase-3 in the execution phase were activated 48h following the H-ras ribozyme treatment. Mitochondrial events characterized by the production of superoxide anion and the release of cytochrome c started at 24h. Mitochondrial transmembrane potential loss occurred 48h after the ribozyme treatment. However, Bcl-2 delayed cytochrome c release to the cytosol, but it could not protect the apoptosis effect, suggesting that cytochrome c release from mitochondria may not play a role in H-ras ribozyme-induced apoptosis.     

Comparison of fumaric acid production by Rhizopus oryzae using different neutralizing agents

Fumaric acid fermentation in a 10-L bubble column fermenter using different neutralizing agents [CaCO(3), Ca(OH)(2), NaHCO(3)] by Rhizopus oryzae ATCC 20344 was examined. It was found that in the fermentation using CaCO(3 )as the neutralizing agent the highest fumaric acid weight yield and volumetric productivity were obtained, 53.4% and 1.03 g/L x h(-1) respectively. In the NaHCO(3) case, the fumaric acid weight yield and volumetric productivity were 33.7% and 0.69 g/L x h(-1), respectively, much lower than the CaCO(3) case. However, the NaHCO(3) alternative has advantages of cell reuse and simple downstream processing because of the high solubility of sodium fumarate. These advantages may offset the disadvantages of using NaHCO(3) as the neutralizing agent, and the overall fumaric acid weight yield and volumetric productivity will increase.     

Mainstream and sidestream cigarette smoke exposure increases Ca2+-dependent phospholipid binding proteins in guinea pig alveolar type II cells

We have earlier identified the presence of a 36 kDa Ca²⁺-dependent phospholipid-binding protein (PLBP) in guinea pig alveolar type II cells. PLBP has been suggested to act as a mediator in facilitating and regulating intracellular surfactant assembly and delivery to the plasma membrane of type II cells for secretion into alveolar space. It has been reported that cigarette smoke exposure (CSE) causes a decrease in the surfactant activity in bronchial washings. We have also reported earlier that mainstream (MS) and sidestream (SS) CSE causes desensitization of -adrenoreceptors in guinea pig alveolar type II cells. Since both Ca²⁺ and -adrenoreceptors are involved in surfactant secretion and PLBP is involved in surfactant delivery, it is important to know whether CSE causes any change in the PLBP level in alveolar type II cells. In the present study, we have demonstrated that MS and SS CSE causes a significant increase in the levels of PLBP in alveolar type II cells (107 and 150%, respectively) and in lung lavage (42 and 125%, respectively) in comparison to that in sham control (430 ng/mg protein in alveolar type II cells and 780 ng/mg protein in lung lavage). The mechanism by which smoke exposure causes an elevation in the levels of PLBP in alveolar type II cells and lung lavage remains to be investigated.     

Crystal structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain

Cytoskeleton-associated proteins (CAPs) are involved in the organization of microtubules and transportation of vesicles and organelles along the cytoskeletal network. A conserved motif, CAP-Gly, has been identified in a number of CAPs, including CLIP-170 and dynactins. The crystal structure of the CAP-Gly domain of Caenorhabditis elegans F53F4.3 protein, solved by single wavelength sulfur-anomalous phasing, revealed a novel protein fold containing three beta-sheets. The most conserved sequence, GKNDG, is located in two consecutive sharp turns on the surface, forming the entrance to a groove. Residues in the groove are highly conserved as measured from the information content of the aligned sequences. The C-terminal tail of another molecule in the crystal is bound in this groove.     

Biomass and aggregation analysis of human embryonic kidney 293 suspension cell cultures by particle size measurement

A method has been developed to monitor the cell growth of aggregated human embryonic kidney 293 (HEK293) suspension cultures by measuring cumulative particle volume and the particle size distribution. This method employs a particle size analyzer that determines the size of individual particles by detecting their light obscuration (blockage) or scattering. Cell counts derived from the cumulative volume of the cell particle correlate well with manual cell counts from a hemacytometer at different stages of growth. This correlation was further confirmed by quantifying total cellular protein of the samples. Simultaneously, the aggregation state of the samples can also be monitored and mathematically described. Results from this study demonstrate that this simple and reproducible method allows the direct measurement of cumulative cell volume and the degree of cell aggregation, as well as an indirect assessment of cell counts.     

Preservation of glucose metabolism in hypertrophic GLUT4-null hearts

GLUT4-null mice lacking the insulin-sensitive glucose transporter are not diabetic but do exhibit abnormalities in glucose and lipid metabolism. The most striking morphological consequence of ablating GLUT4 is cardiac hypertrophy. GLUT4-null hearts display characteristics of hypertrophy caused by hypertension. However, GLUT4-null mice have normal blood pressure and maintain a normal cardiac contractile protein profile. Unexpectedly, although they lack the predominant glucose transporter in the heart, GLUT4-null hearts transport glucose and synthesize glycogen at normal levels, but gene expression of rate-limiting enzymes involved in fatty acid oxidation is decreased. The GLUT4-null heart represents a unique model of hypertrophy that may be used to study the consequences of altered substrate utilization in normal and pathophysiological conditions.     

Protein coding gene trees in ciliates: comparison with rRNA-based phylogenies

We have reexamined the phylogeny of the ciliates using alpha-tubulin and phosphoglycerate kinase gene sequences. For alpha-tubulin, we have compared the amino acid and nucleotide sequences of 20 species representing seven of the nine classes of the phylum (Karyorelictea, Heterotrichea, Hypotrichea, Oligohymenophorea, Colpodea, Nassophorea, and Litostomatea). The phylogenetic tree resembles a bush from which three monophyletic lineages can be distinguished which correspond to the three classes Hypotrichea, Oligohymenophorea, and Litostomatea. For phosphoglycerate kinase, we have compared the amino acid sequences from 7 species representing three classes (Heterotrichea, Hypotrichea, and Oligohymenophorea). The branching pattern is resolved in three deeply separated branches with an early emergence of the heterotrich. Our comparative analysis shows that if alpha-tubulin phylogeny is not informative at the interclass level, the preliminary data from the phosphoglycerate kinase molecule appear more promising. Nevertheless, at low taxonomic level and at the class level, the resolved phylogenetic relationships inferred from both protein and rRNA sequence data are congruent.     

High temperature slows down growth in tobacco hornworms (Manduca sexta larvae) under food restriction

When fed ad libitum (AL), ectothermic animals usually grow faster and have higher metabolic rate at higher ambient temperature. However, if food supply is limited, there is an energy tradeoff between growth and metabolism. Here we hypothesize that for ectothermic animals under food restriction (FR), high temperature will lead to a high metabolic rate, but growth will slow down to compensate for the high metabolism. We measure the rates of growth and metabolism of 4 cohorts of 5th instar hornworms (Manduca sexta larvae) reared at 2 levels of food supply (AL and FR) and 2 temperatures (20 and 30 °C). Our results show that, compared to the cohorts reared at 20 °C, the ones reared at 30 °C have high metabolic rates under both AL and FR conditions, but a high growth rate under AL and a low growth rate under FR, supporting this hypothesis.