Livin and caspase-3 expression are negatively correlated in cervical squamous cell cancer.

OBJECTIVE: Overexpression in cancer cells of inhibitor of apoptosis proteins like livin appears to promote tumorigenesis by regulating expression of proteins involved in apoptosis signaling. Here, the authors investigated expression of livin and an apoptosis protein that is known to inhibit, caspase-3, in cervical squamous cell carcinoma. MATERIALS AND METHODS: Their expression was assessed for correlation with tumor invasiveness. Immunohistochemistry for livin and caspase-3 was used in 36 normal cervical tissues and in 98 samples of cervical squamous cell carcinoma. The percentage of cells expressing these proteins was compared between normal and cancer samples. Their expression rates in cancer samples were subsequently compared with one another and with the clinical and pathological characteristics of the samples. RESULTS: Livin was more commonly expressed in tumor samples than in normal tissues, while the opposite pattern was observed for caspase-3. Expression of livin was significantly associated with advanced clinical stage, higher pathological grade, and lymph node metastasis (p < 0.05). Expression of caspase-3 was significantly associated with lower clinical stage, lower pathological grade, and lack of lymph node metastasis (p < 0.05). Finally, expression of livin was negatively correlated to caspase-3 expression in cervical squamous cell carcinoma tissue (r = -0.57, p < 0.05). CONCLUSIONS: Livin may inhibit apoptosis in cervical squamous cell carcinoma by downregulating caspase-3, thereby promoting disease progression.

The detection of 3 leptin receptor isoforms in crucian carp gill and the influence of fasting and hypoxia on their expression.

To understand leptin signaling pathway in the crucian carp (Carassius carassius), we cloned 3 leptin receptor isoform complementary DNAs (ie, the long form [cclpr-L], the short form [cclpr-s1], and the secreted form [cclpr-s2]). Variant cclpr-L had a 3,255-bp open reading frame and a complete intracellular domain with box 1 and box 2 consensus sequences. By contrast, cclpr-s1 contained only 4 amino acids in its intracellular domain, without the "box 1" motif, which is conserved among membrane-bound leptin receptor short isoforms in mammals. Variant cclpr-s2 had no transmembrane domain, suggesting that it is a soluble form of the receptor, and alternative splicing of cclpr-s2 mRNA employs a different mechanism for the generation of soluble leptin receptor by intron retention. The fasting-treated fish showed significantly lower cclpr-L mRNA levels in gill tissue than the control group, whereas cclpr-s2 mRNA levels did not vary significantly among the groups. Treatment with hypoxia significantly increased mRNA levels of both cclpr-L and cclpr-s2 in gill tissue. To our knowledge, this is the first study of leptin receptor isoforms expression in teleosts.

A 16-bp RBE element mediated Rep-dependent site-specific integration in AAVS1 transgenic mice for expression of hFIX.

We earlier reported that, a 16 bp Rep-binding element (RBE) was sufficient for mediating Rep-dependent integration into AAVS1 in vitro. We explored here the potential use of this RBE in site-specific genome integration at the AAVS1 site in vivo using transgenic mice carrying the human AAVS1 locus in their genome. In the presence of a Rep-donor plasmid, an human blood coagulation factor IX (hFIX) expression plasmid (pRBE-CMV-hFIX) containing the 16 bp RBE was delivered to AAVS1 transgenic mice by hydrodynamic injection. Insertion of the transgene into the AAVS1 site of the mouse genome was confirmed by nested PCR at the junction of the plasmid/AAVS1 locus. Sequencing analysis found the site-specific insertion in four of seven animals injected with pRBE-CMV-hFIX but in none of the mice injected with pN2-CMV-hFIX, the control construct without the 16 bp RBE or with pRBE-CMV-hFIX plasmid but without co-expressing Rep. Plasma hFIX levels in pRBE-CMV-hFIX-injected animals were higher and lasted longer than in the pN2-CMV-hFIX control group. The levels of hFIX in pRBE-CMV-hFIX-injected animals were also significantly higher than in the control animals after partial hepatectomy (PH). These results showed that the 16 bp RBE could mediate the delivery of a therapeutic gene into the AAVS1 locus in a Rep-dependent, site-specific manner in vivo, suggesting its potential application in gene therapy.

The synergistic activity between Cry1Aa and Cry1c from Bacillus thuringiensis against Spodoptera exigua and Helicoverpa armigera.

AIMS: To investigate the interaction between two crystal proteins, Cry1Aa and Cry1C, for future development of biopesticides based on Bacillus thuringiensis, toxicities of the two individual proteins and in combinations have been determined against Spodoptera exigua and Helicoverpa armigera larvae, and synergism between the proteins has been evaluated using synergistic factor. METHODS AND RESULTS: SDS-PAGE showed that Cry1Aa and Cry1C proteins could be expressed in acrystalliferous B. thuringiensis 4Q7 strain, with molecular weights of 135 and 130 kDa respectively. The bioassay results indicated a synergistic activity between Cry1Aa and Cry1C against S. exigua and H. armigera, and the highest toxicities could be observed in the combination of Cry1Aa and Cry1C at a ratio of 1 : 1. CONCLUSION: The two toxins, Cry1Aa and Cry1C, interact synergistically to exhibit higher toxicity against S. exigua and H. armigera. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the investigation on the synergistic activity between two B. thuringiensis Cry1 toxins. It can be applied to the rational design of new generations of B. thuringiensis biopesticides and to strategies for management of resistant insects.

Uterine secretion from mares with post-breeding endometritis alters sperm motion characteristics in vitro.

Uterine secretion was collected from five normal mares during estrus by the use of a tampon. In subsequent estrus cycles, mares were inseminated with 1 x 10(9) spermatozoa from a stallion of known fertility, and uterine secretion was collected randomly at 6, 12, and 24 hours after insemination. All mares had negative endometrial cytology before insemination. At the time of uterine secretion sampling, semen was collected from two stallions and extended with Kenney's extender to a concentration of 50 x 10(6) spermatozoa/mL. Extended semen was diluted 2:1 with uterine secretion; semen extender; and centrifuged uterine secretion (noncellular). Samples were kept at room temperature and sperm motion characteristics (corrected motility (CMOT), progressively motile spermatozoa (PMS), and mean path velocity (MPV) were evaluated using a computer-assisted semen analyzer every 40 minutes for a total of 4 hours. Sperm motion characteristics of spermatozoa were significantly better when incubated in semen extender compared to uterine secretion (P < 0.05). The CMOT and PMS were significantly better in uterine secretion collected before, compared to after AI with the lowest values observed in samples collected at 12 hours after breeding (P < 0.05). Sperm motion characteristics of spermatozoa incubated in centrifuged uterine secretion was only slightly suppressed compared to spermatozoa incubated in semen extender, suggesting that the altered motion characteristics were mostly due to the presence of polymorphonuclear neutrophils (PMNs) in the samples. It was concluded from this study that spermatozoa can survive in inflamed uterine secretion, but that sperm motion characteristics in vitro are altered.

Effect of ration and exercise on plasma creatine kinase activity and lactate concentration in Thoroughbred horses with recurrent exertional rhabdomyolysis.

OBJECTIVE: To determine the effects of 3 rations (low grain, fat, high grain) on plasma creatine kinase (CK) activity and lactate concentration in Thoroughbred horses with recurrent exertional rhabdomyolysis (RER). ANIMALS: 5 Thoroughbreds with RER and 3 healthy Thoroughbreds (control horses). PROCEDURES: Rations were formulated to meet (low-grain and fat rations) or exceed (high-grain ration) daily energy requirements. Each ration was fed to horses in a crossover design for 3 weeks. Horses were exercised on a treadmill Monday through Friday; maximum speed on Monday and Friday was 11 m/s (6% slope), on Tuesday and Thursday was 9 m/s, and on Wednesday was 4.5 m/s. Plasma CK activity and lactate concentration were determined before and after exercise. RESULTS: Horses with RER fed the high-grain ration had significantly greater CK activity and change in CK activity 4 hours after exercise, compared with those fed the low-grain ration. Horses with RER exercised at the trot or canter had significantly greater increases in CK activity, compared with those exercised at the gallop. Plasma lactate concentrations after exercise were similar in control and affected horses. Lactate concentration and CK activity were not correlated in horses with RER. CONCLUSIONS AND CLINICAL RELEVANCE: Rations high in grain and formulated to exceed daily energy requirements may increase episodes of rhabdomyolysis in thoroughbred horses susceptible to RER.

[Study on expression and resistance of the double drug resistance genes transduced into human umbilical cord blood CD34+ cells mediated by bicistronic retroviral vector].

To explore whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH1) and multidrug resistance gene (MDR1) increase resistance to 4-Hyaroxycyclophosphophamide (4-HC) and P-Glycoprotein Effluxed Drugs, a bicistronic Retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed. The vector was transduced into the packaging cell lines GP + E86 and PA317 by LipofectAMINE. Using the medium containing VCR and 4-HC for cloning selection and pingponging supernatant infection between ecotropic producer clone and amphotropic producer clone, we obtained high titer amphotropic PA317 producer clone with the highest titer up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfectced repeatedly with supernatant of retrovirus containing human ALDH1 and MDR1cDNA under stimulation of hemopoietie growth factors. PCR, RT-PCR, Southern blot, Northern blot, FACS and MTT method analyses show that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4- to 7.2-folds stronger resistance to cyclophospsphamede and P-Glycoprotein Effluxes drug in comparison with the nontransduced cells. This study provided a foundation for the application of combination chemotherapy in tumor clinical trial.

Long-term results of intratumorous bleomycin-A5 injection for head and neck lymphangioma.

From June 1987 to June 1995, 200 patients with various types of head and neck lymphangioma were treated by intratumorous injection of bleomycin-A5. All 200 cases were followed from 2 to 10 years; 95 cases were followed for more than 5 years. The effective rate was 97%, and the curative rate was 86.5%. No pulmofibrosis or other serious complications were found. The relationships between the curative effect and tumor type, size, and side effect of bleomycin are discussed.

[Splicing and stability of intron in the expression retroviral vector with human clotting factor IX].

To study the role of intron in the expression of hFIX, retroviral vectors with intron containing hFIX were constructed. It is fundamental for the intron study whether the intron constructed in retroviral vector can be steadily transferred into target cell. First, we constructed two forward-orientation retroviral vectors: G1NaC-i-IX contains the exogenous intron from IL-2, and G1NaC-i'-IX contains the truncated intron I from hFIX gene, covering the splicing donor and acceptor sequences. RT-PCR result indicated that intron in the forward-orientation retroviral vector was spliced after packaging in PA317. Then, reverse-orientation retroviral vectors G1NaC-i'-IXR and G1NaPAIXi' BAM were constructed, in which the reverse and complimentary sequences of hFIX gene with intron appeared in retroviral RNA. RT-PCR assay combined with ELISA test indicated that intron was retained after packaging and hFIX gene with intron constructed in the reverse-orientation retroviral vector can be transduced intact and expressed hFIX at a high level in vitro.

Glucose tolerance, luteinizing hormone release, and reproductive performance of first-litter sows fed two levels of energy during gestation.

This experiment was conducted to determine the effects of gestational energy intake on glucose tolerance, LH profiles, and reproductive performance of first-litter sows. Sixteen pregnant gilts were assigned to either high energy (11 Mcal of ME/d; H) or normal energy (6.5 Mcal of ME/d; N) diets during gestation. They did not receive the treatment diets until 35 d of gestation and had free access to the same commonly used diet during lactation. A glucose tolerance test (1 g glucose per kg BW) was conducted after 18 h of feed deprivation on 110 d of gestation and 15 d of lactation. Blood samples were collected at -10, -5, 0 (immediately before glucose infusion), 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 150, and 180 min for assay of glucose and insulin concentrations. Blood samples were also taken on d 7, 14, and 21 of lactation and on 1 d postweaning for 6 h with 10-min intervals for LH characteristics. Sows in Group H had greater gain of BW and backfat during gestation (P < .001) and had less feed intake and greater weight loss during lactation (P < .002) than those in Group N. Sows in Group H (8.0 d) showed 1.6 d longer (P = .07) weaning-to-estrus intervals than those in Group N (6.4 d). After glucose infusion, insulin levels in Group H were higher (P < .05) than those in Group N. Lower LH concentrations on 1 d postweaning were found (P < .05) in sows in Group H than in Group N. This study indicates that energy intake during gestation is negatively related to feed intake during lactation. Reduced feed intake during lactation of sows having high gestational energy impairs glucose tolerance, suggesting that interaction of insulin with LH secretion may influence weaning-to-estrus intervals.