[Correlation between serum growth differentiation factor 11 level and severity of coronary artery disease in patients with acute myocardial infarction].

Objective: To investigate the correlation between serum growth differentiation factor 11 (GDF11) level and coronary artery lesions in patients with ST-segment elevation myocardial infarction (STEMI), and the predictive efficacy of nomogram risk prediction model based on GDF11 combined with traditional risk factors on the occurrence of STEMI. Methods: This study was a retrospective cross-sectional study. Patients hospitalized in the Department of Cardiology of the 904th Hospital of Joint Logistic Support Force of People's Liberation Army of China from 2016 to 2018 were selected and divided into control group and STEMI group. The demographic data, blood lipid level, laboratory indicators of blood and GDF11 level were collected. Logistic regression analysis screened out independent correlated factors for the occurrence of STEMI. Spearman correlation analysis clarified the correlation of each indicator with the SYNTAX or Gensini scores. A nomogram risk prediction model for the risk of STEMI occurrence and the receiver operating characteristic curve was used to compare the prediction efficiency of each model. Results: A total of 367 patients were enrolled, divided into control group (n=172) and STEMI group (n=195), age (66.5±11.8), male 222 (60.49%). The serum GDF11 level of STEMI group was significantly lower than that of the control group (36.20 (16.60, 70.75) µg/L vs. 85.00 (53.93, 117.10) µg/L, P<0.001). The results of multivariate logistic regression analysis showed serum GDF11(OR=0.98, 95%CI: 0.97-0.99) and traditional independent risk factors such as smoking, diabetes, C-reactive protein, homocysteine, lipoprotein (a) and apolipoprotein A1/B were independent correlate factors for the occurrence of STEMI (P<0.05). Spearman correlation analysis showed that serum GDF11 was negatively correlated with SYNTAX score and Gensini score (P<0.05). The nomogram model constructed by serum GDF11 combined with traditional independent risk factors (AUC=0.85, 95%CI: 0.81-0.89) had better predictive value for the occurrence of STEMI than the traditional nomogram model constructed by independent risk factors(AUC=0.80, 95%CI:0.75-0.84) or serum GDF11 (AUC=0.76, 95%CI: 0.72-0.81), all P<0.01. Conclusions: Serum GDF11 is an independent correlate factor in the occurrence of STEMI and is negatively correlated with the severity of coronary artery lesions in patients with STEMI. The nomogram model constructed based on GDF11 combined with traditional risk factors can be a good predictor for the occurrence of STEMI.

[The role and mechanism of human trophoblastic cell surface antigen 2 in the invasion and metastasis of ovarian cancer].

Objective: To explore the role and molecular mechanism of trophoblastic cell surface antigen 2 (Trop2) in the invasion and migration of ovarian cancer. Methods: Through the data mining of Cancer Cell Line Encyclopedia and TCGA database, the clinical significance of Trop2 expression was analyzed. Western blot was used to detect Trop2 protein expression in ovarian cancer cell lines including A3O, A1780 and SKOV3. SKOV3 cells were used to construct Trop2-short hairpin RNA (shRNA) cell model. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to detect the SKOV3 mRNA expression in SKOV3-shRNA and SKOV3-NC cells. Cell counting kit-8 (CCK8) was used to detect the proliferation of SKOV3-shRNA cells and SKOV3-NC cells. Flow cytometry was used to detect cell cycle and apoptosis in two groups of cells. Transwell array was used to detecte the invasion and migration of SKOV3-shRNA cells and SKOV3-NC cells. Western blot was used to detect the protein expressions of AKT, p-AKT, ß-catenin, caspase3, bcl-2, E-cadherin and vimentin. Results: Trop2 mRNA highly expressed in ovarian cancer, and was related to the tumor stage and patient survival. Compared with A3O cells, Trop2 overexpressed in A1780 and SKOV3 cells (P<0.05). The relative expression levels of Trop2 mRNA in SKOV3-NC group and SKOV3-shRNA group were 1.18±0.24 and 0.42±0.08, with statistically significant difference (P<0.05). The results of CCK-8 array showed that the cell viability of SKOV3-NC group was significantly higher than that of SKOV3-shRNA group (P<0.05). The proportion of G(0)/G(1) cells in SKOV3-NC and SKOV3-shRNA groups were (38.67±4.22)% and (60.24±8.17)%, respectively. G(0)/G(1) arrest was observed in SKOV3-shRNA cells (P<0.05). The apoptosis rate of SKOV3-shRNA group was (26.32±1.81)%, significantly higher than (6.54±1.32)% of SKOV3-NC group (P<0.05). The number of migrating SKOV3 cells in the SKOV3-shRNA and SkOV3-NC groups were 1 255.83±108.44 and 1 679.71±213.92, while the number of invading cells were 242.49±52.09 and 473.54±73.11, respectively. Compared with the SKOV3-NC group, the number of migrating and invading SKOV3-shRNA group was significantly reduced (all P<0.05). The expressions of p-AKT2, Bcl-2, vimentin and ß-catenin were down-regulated, and the expressions of caspase 3 and E-cadherin were up-regulated in SKOV3-shRNA cells. There was no significant change in the total protein level of AKT. Conclusions: Trop2 expression is related to ovarian cancer stage and postoperative survival. Trop2 can promote ovarian cancer cell proliferation and metastasis by activating the AKT/ß-catenin signaling pathway and knockdown of Trop2 inhibits the progression of ovarian cancer.

Dietary supplementation with arginine and glutamic acid alters the expression of amino acid transporters in skeletal muscle of growing pigs.

Sixty Duroc × Large White × Landrace pigs with an average initial body weight (BW) of 77.1 ± 1.3 kg were selected to investigate the effects of dietary supplementation with arginine (Arg) and/or glutamic acid (Glu) on free amino acid (FAA) profiles, expression of AA transporters, and growth-related genes in skeletal muscle. The animals were randomly assigned to one of five treatment groups (basic diet, iso-nitrogenous, Arg, Glu, and Arg + Glu groups). The results showed that plasma Glu concentration was lowest in the Arg + Glu group and highest in the Glu group (P < 0.05). In the longissimus dorsi (LD) muscle, the concentrations of histidine, Arg, and taurine in the Arg + Glu group were higher, and the concentrations of 3-methylhistidine was lower, than in the basic diet group (P < 0.05). The mRNA levels of ASC amino acid transporter-2 (ASCT2), L-type AA transporter 1, and sodium-coupled neutral amino acid transporter 2 in the LD muscle, as well as the mRNA levels of ASCT2 and proton-assisted amino acid transporter in the biceps femoris (BF) muscle, were higher in the Arg + Glu group compared to the basic diet group (P < 0.05). The mRNA levels of the muscle-specific RING finger-1 and muscle atrophy F-box genes in the LD muscle were downregulated in the Glu and Arg + Glu groups compared to the basic diet group (P < 0.05). Collectively, these findings suggest that dietary supplementation with both Arg and Glu increases intramuscular FAA concentrations and decreases the mRNA levels of genes involved in protein degradation in skeletal muscle.

Association between the XPG gene Asp1104His polymorphism and lung cancer risk.

It has been suggested that the xeroderma pigmentosum complementation group G (XPG) gene Asp1104His polymorphism is linked to susceptibility to lung cancer. However, the results from the published studies are contradictory rather than conclusive. With this meta-analysis, we aimed to achieve a better understanding of the effects of the XPG gene Asp1104His polymorphism on lung cancer risk. We identified six eligible studies from five publications that included a total of 2293 lung cancer patients and 2586 controls. There was a significant association between the XPG gene Asp1104His polymorphism and lung cancer (His/His vs Asp/Asp: OR = 1.24, 95%CI = 1.04-1.48; Asp/His vs Asp/Asp: OR = 1.17, 95%CI = 1.03-1.34; the dominant model: OR = 1.18, 95%CI = 1.04-1.33; the recessive model: OR = 1.10, 95%CI = 0.94-1.28). In a subgroup analysis by nationality, we found a significant association between the XPG gene Asp1104His polymorphism and lung cancer risk in Asians. No publication bias was found in this study. The results from this meta-analysis indicate that the XPG gene Asp1104His polymorphism is associated with lung cancer risk, especially in Asians.

[Recent advances and some consideration about the trans-oral laporascopic thyroidectomy].

Trans-oral laporascopic thyroidectomy is a innovative technique that incoporated the concept of natural orifice transluminal endoscopic surgery. Compare to other endoscopic thyroidectomy, trans-oral laporascopic thyroidectomy is characterised with no scar in the body surface, better cosmetic results and conforms to the concept of minimally invasive surgery. At present, trans-oral laporascopic thyroidectomy is still in the initial stages of clinical application and gains a great deal of attention from surgeons all over the world. It mainly includes oral bottom approach and oral vestibular approach. Proponents and researchers in this field have been attempting to use this technique for treatment of thyroid cancer and combine it with the robotics assisted surgical system. In the future, the indications and contraindications of trans-oral laporascopic thyroidectomy need to be further studied and evaluated. Complications and surgical procedures still have many issues need to be solved.

Apparent diffusion coefficient measurements of bilateral kidneys at 3 T MRI: effects of age, gender, and laterality in healthy adults.

AIM: To investigate the effects of age and gender on apparent diffusion coefficient (ADC) measurements of bilateral kidneys at 3 T MRI, and compare the ADC values of left and right kidneys. MATERIALS AND METHODS: In all, 137 healthy participants (mean age 42.8 ± 14.7 years; age range 16-75 years) comprising 68 male and 69 female participants were enrolled. Three Tesla echo-planar diffusion-weighted imaging (DWI) of bilateral kidneys was performed and ADC values were measured in the cortex, medulla, and whole parenchyma. Pearson correlation analysis and linear regression were performed to determine the associations between the ADC values in each region and age. Effects of age and gender on ADC values were analysed using two-factor analysis of variance (ANOVA). The paired-samples t-test was established to compare the ADC values between left and right kidneys. RESULTS: ADC values were significantly higher in the young group (≤50 years) than in the old group (>50 years), and correlated inversely with the age in all regions. Male participants had higher ADC values than female participants in all regions except left medulla. Two-factor ANOVA of age × gender showed no significant interactions between the variables age and gender were found. No significant differences in ADC values between left and right kidneys were observed. CONCLUSION: Renal ADC values are age- and gender-dependent, and show no significant difference between left and right kidneys. Age- and gender-related effects should be taken into consideration in future renal DWI studies when using normal ADC values from health controls.

Effects of dietary L-arginine or N-carbamylglutamate supplementation during late gestation of sows on the miR-15b/16, miR-221/222, VEGFA and eNOS expression in umbilical vein.

Placental vascular formation and blood flow are crucial for fetal survival, growth and development, and arginine regulates vascular development and function. This study determined the effects of dietary arginine or N-carbamylglutamate (NCG) supplementation during late gestation of sows on the microRNAs, vascular endothelial growth factor A (VEGFA) and endothelial nitric oxide synthase (eNOS) expression in umbilical vein. Twenty-seven landrace×large white sows at day (d) 90 of gestation were assigned randomly to three groups and fed the following diets: a control diet and the control diet supplemented with 1.0% L-arginine or 0.10% NCG. Umbilical vein of fetuses with body weight around 2.0 kg (oversized), 1.5 kg (normal) and 0.6 kg (intrauterine growth restriction, IUGR) were obtained immediately after farrowing for miR-15b, miR-16, miR-221, miR-222, VEGFA and eNOS real-time PCR analysis. Compared with the control diets, dietary Arg or NCG supplementation enhanced the reproductive performance of sows, significantly increased (P<0.05) plasma arginine and decreased plasma VEGF and eNOS (P<0.05). The miR-15b expression in the umbilical vein was higher (P<0.05) in the NCG-supplemented group than in the control group. There was a trend in that the miR-222 expression in the umbilical vein of the oversized fetuses was higher (0.05

[The textbooks in the North China Medical College].

The North China Medical College typically represented medical colleges for traditional Chinese medicine in the 1930s when many of them were set up. It was based on the principles of centring on traditional Chinese medicine, following western medicine and integrated medicine in teaching. This led to the emergence of a great number of people with a high level of traditional Chinese medicine and strong belief in it. In terms of the textbooks and handouts for western medicine, compared to similar textbooks in other medical colleges, such as the Medical College of Xie He, at that time, the textbooks in the North China Medical College covered a variety of perspectives and categories. It was found that 20 textbooks for western medicine in the North China Medical College were designed reasonably in content and were simple and applicable in teaching. More importantly, it contained some traditional Chinese medicine in different degrees, with its typical characteristics. The course design and textbook compilation provided references for the teaching in contemporary medical universities.

The presence and expression of the hepatitis B virus in human oocytes and embryos.

BACKGROUND: The objective of this study was to explore the potential for vertical transmission of hepatitis B virus (HBV) from parents to offspring via human germ cells. METHODS: For study samples, 250 oocytes from hepatitis B surface antigen (HBsAg) seropositive women and 578 embryos from couples with at least one HBsAg seropositive partner were collected. HBV DNA in the nuclei of oocytes and embryos was detected by fluorescence in situ hybridization; HBsAg expression was analysed using immunofluorescence; and serum HBV DNA levels were measured by real-time PCR. The HBV infection duration of the women and the serum HBsAg status of their mothers were also examined. RESULTS: HBV DNA was present in 9.6% (24/250) of oocytes and 14.4% (83/578) of embryos. Rates of HBV DNA positive embryos were similar among couples in which the woman, man or both partners were HBsAg seropositive, 13.1% (57/436), 21.3% (16/75) and 14.9% (10/67), respectively. Rates of positivity in oocytes and embryos were significantly higher in a group with high serum levels HBV DNA than in a group with lower serum levels (P= 0.004 and P= 0.002, respectively). Higher rates of oocyte positivity were found for women whose mothers were HBV infected compared with those with uninfected mothers. Expression of HBsAg was observed in 8.7% (2/28) oocytes and 14.1% (10/71) embryos (P= 0.34). CONCLUSIONS: The presence of HBV DNA in human oocytes or embryos was related to the women's serum levels of HBV DNA and the infection status of their mothers. The HBV positive embryos were either maternally or paternally dependent. HBV infection may result in vertical transmission to the offspring via germ cells.

The effects of dexmedetomidine administered at various times on acute lung injury in rats.

OBJECTIVE: To investigate the effects of dexmedetomidine (Dex) treatment administered at various times on acute lung injury (ALI). MATERIALS AND METHODS: Thirty Wistar rats were randomly divided into five groups (n = 6/group). Lipopolysaccharide (LPS) was intraperitoneally injected into the rats in the LPS, Dex1, Dex2, and Dex3 groups to induce ALI, while the control group (C) was left untreated. Rats in the Dex1 group were intraperitoneally administered with 50 µg/kg Dex 30 minutes before modeling. Rats in the Dex2 group were injected with 25 µg/kg Dex 30 minutes before modeling and two hours after. Rats in the Dex3 group received 50 µg/kg Dex two hours after modeling. The animals in the C and LPS groups were given an equal volume of saline. The wet-to-dry (W/D) weight ratio of the rats' lungs was calculated, and pathological alterations in lung tissues were observed. The concentrations of inflammation-related factors and the expression of Janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), and matrix metallopeptidase 9 (MMP9) were measured. RESULTS: The W/D ratio, expression of inflammatory factors, and expression of JAK1, STAT3, and MMP9 were significantly increased in the ALI rats (p < 0.05) compared with the C group. The level of anti-inflammatory factors in the Dex-treated groups was also significantly increased compared with the LPS group (p < 0.05). The concentration of anti-inflammatory factors in the Dex2 group was significantly higher than that recorded in the Dex1 and Dex3 groups (p < 0.05). CONCLUSIONS: Dex treatment administered at different times protects rats against LPS-induced ALI to varying degrees. The protective effects of Dex were most robust when administered both before and after LPS stimulation.

[Endoscopic lateral neck dissection via the breast and transoral approaches for papillary thyroid carcinoma: a report of 10 cases].

Objective: To investigate the feasibility of endoscopic lateral neck dissection via the breast and transoral approaches (ELNDBTOA) for papillary thyroid carcinoma (PTC). Methods: From February 2015 to April 2019, 10 patients with PTC (cN1b) including 1 male and 9 females aged from 22 to 53 years old received ELNDBTOA in the General Surgery Department of Zhongshan Hospital, Xiamen University. Total thyroidectomy, the central lymph node dissection and the selective neck dissection (levels Ⅱ, Ⅲ and Ⅳ) were performed endoscopically via the breast approach, and then the residual lymph nodes were dissected via transoral approach. The medical records, operation time, blood loss, complications and postoperative follow-up outcomes were analyzed retrospectively. SPSS 22.0 software package was used for statistical processing of clinical data of patients. Results: All cases were successfully treated with ELNDBTOA without transfer to open surgery. The average operative time was (362.5±79.7) min, the blood loss was (23.0±14.9) ml, and the postoperative hospital stay was (5.1±1.3) days. The mean number of harvested cervical lymph nodes were (34.2±25.8), and the mean number of positive lymph nodes were (6.5±4.9). Lymph nodes were dissected by the further dissection via oral approach in 6 patients and a total of 9 lateral lymph nodes were havested from 2 of the 6 patients, with 3 positive lymph nodes. Two patients had transient skin numbness in the mandibular area and recovered within two weeks. One patient developed transient hypoparathyroidism and recovered within two months. No secondary bleeding, recurrent laryngeal nerve paralysis, chylous leakage, neck infection, permanent hypoparathyroidism or other complications were observed. The follow-up time was from 16 to 66 months with a median of 42.5 months, no tumor recurrence or metastasis occurred, and also no obvious deformity, abnormal sensation or movement in the chest, neck and mouth was observed. Conclusions: ELNBTOA is safe and feasible, with good cosmetic outcome.

[Clinical experience in transoral endoscopic thyroidectomy via sublingual and vestibular approach: a report of 98 cases].

Objective: To summarize the clinical experience in transoral endoscopic thyroidectomy via sublingual and vestibular approach (TOETSLVA). Methods: We retrospectively reviewed the medical record of patients who underwent TOETSLVA in our department from November 2011 to May 2020, including 3 males and 95 females, aged from 18 to 57 years old. Initial 81 cases were categorized in "Period A (November 2011-November 2015)" and subsequent 17 cases in "Period B (August 2019-May 2020)" . Data about demographics, operation time and complications were collected. SPSS 22.0 software was used for statistical analysis. Results: In Period A, the average age of patients was (34.2±9.4) years old; the mean tumor diameter was (2.33±0.80) cm; postoperative pathology showed benign nodules in 76 cases and malignant carcinoma in 5 cases; there were 65 cases of unilateral subtotal thyroid lobectomy, 6 cases of isthmus lobectomy, and 5 cases of bilateral subtotal thyroid lobectomy, with the mean operation time of (132.70±47.22) min; in 5 cases of unilateral thyroid lobectomy with central lymph node dissection, the mean operation time was (185.4±31.40) min; postoperative neck infections occurred in 6 cases; temporary vocal cord paralysis occurred in 1 case, which it recovered within two months; and CO(2) gas embolism occurred in 2 cases. In Period B, the average age of patients was (35.1±8.5) years old; mean tumor diameter was (1.32±0.67) cm; postoperative pathology indicated malignant nodules in 15 cases and benign nodules in 2 cases; in 2 cases of unilateral thyroid lobectomy, the mean operation time was (153.5±34.64) min; in 15 cases of unilateral thyroid lobectomy with central lymph node dissection, the mean operation time was (123.73±14.26) min; and none of patients developed postoperative neck infections or CO(2) gas embolism. All patients had different degree of cutaneous numbness in the submandibular region after surgery, which recovered within 1-2 weeks. There were no complications such as postoperative secondary hemorrhage, permanent vocal cord paralysis, hypoparathyroidism in both the periods. The median follow-up time was 86 months (57-105 months) in Peroid A and 5 months (3-12 months) in Peroid B. During the follow-up periods, there were no obvious abnormalities in swallowing, chewing, oral sensory function and neck activity, and also no tumor recurrence or metastasis. Conclusions: TOETSLVA is a safe and feasible surgery method, with a good cosmetic result. This approach will not lead to a postoperative cutaneous numbness of the submandibular region for a long time.

Dietary supplementation with Astragalus polysaccharide enhances ileal digestibilities and serum concentrations of amino acids in early weaned piglets.

Two experiments were conducted to evaluate the effects of dietary supplementation with Astragalus polysaccharide (APS) on growth performance, apparent ileal digestibilities (AID) of amino acids (AA), and their serum concentrations in early weaned piglets. In Exp. 1, 60 pigs were weaned at 21 days of age (BW 7.35 +/- 0.23 kg) and allocated to three treatments (20 pigs/treatment), representing supplementing 0.0% (control), 0.02% colistin (antibiotic), or 0.1% APS to a corn- and soybean meal-based diet. Average daily gain (ADG), average daily feed intake (ADFI), and feed/gain ratio (F/G) were measured weekly. Blood samples were obtained from five pigs selected randomly from each treatment for the measurement of serum free AA concentrations on days 7, 14, and 28. In Exp. 2, 12 pigs were weaned at 21 day of age (BW 7.64 +/- 0.71 kg), assigned to three treatment groups as in Exp. 1, and surgically fitted with a simple T-cannula at the terminal ileum. Ileal digesta samples were obtained for the measurement of AID of AA on days 7, 14 and 28. Dietary APS did not affect ADFI, but enhanced (P < 0.05) ADG by 11 and 4.4%, and improved F/G by 5.6 and 8.4%, respectively, compared with the control and antibiotic groups. Addition of APS to the diet increased AID and serum concentrations of most nutritionally essential and non-essential AA (including arginine, proline, glutamate, lysine, methionine, tryptophan, and threonine) on days 14 and 28. Circulating levels of total AA were affected by the age of pigs and treatment x time interaction. Collectively, these findings indicate that APS may ameliorate the digestive and absorptive function and regulate AA metabolism to beneficially increase the entry of dietary AA into the systemic circulation, which provide a mechanism to explain the growth-promoting effect of APS in early weaned piglets.

Promotion of dendritic growth by CPG15, an activity-induced signaling molecule.

Activity-independent and activity-dependent mechanisms work in concert to regulate neuronal growth, ensuring the formation of accurate synaptic connections. CPG15, a protein regulated by synaptic activity, functions as a cell-surface growth-promoting molecule in vivo. In Xenopus laevis, CPG15 enhanced dendritic arbor growth in projection neurons, with no effect on interneurons. CPG15 controlled growth of neighboring neurons through an intercellular signaling mechanism that requires its glycosylphosphatidylinositol link. CPG15 may represent a new class of activity-regulated, membrane-bound, growth-promoting proteins that permit exquisite spatial and temporal control of neuronal structure.

Stabilization of dendritic arbor structure in vivo by CaMKII.

Calcium-calmodulin-dependent protein kinase II (CaMKII) promotes the maturation of retinotectal glutamatergic synapses in Xenopus. Whether CaMKII activity also controls morphological maturation of optic tectal neurons was tested using in vivo time-lapse imaging of single neurons over periods of up to 5 days. Dendritic arbor elaboration slows with maturation, in correlation with the onset of CaMKII expression. Elevating CaMKII activity in young neurons by viral expression of constitutively active CaMKII slowed dendritic growth to a rate comparable to that of mature neurons. CaMKII overexpression stabilized dendritic structure in more mature neurons, whereas CaMKII inhibition increased their dendritic growth. Thus, endogenous CaMKII activity limits dendritic growth and stabilizes dendrites, and it may act as an activity-dependent mediator of neuronal maturation.

The use of surrogate vascular markers in youth at risk for premature cardiovascular disease.

Premature cardiovascular disease (CVD) begins in youth--a crucial period when modification of the disease may have the greatest impact. Failure to diagnose preclinical CVD at this stage misses a major opportunity to prevent the long-term consequences of this disease. An array of surrogate vascular markers (SVMs) are now available that can determine the extent of preclinical vascular injury in the pediatric population. These SVMs include flow-mediated vasodilatation, carotid intima media thickness, arterial stiffness, and biomarkers including high sensitivity C-reactive protein, cell adhesion molecules and methylarginines. We believe that the use of these SVMs will help to develop a better understanding of early pathological vascular changes in youth, facilitate earlier diagnosis of preclinical atherosclerosis and provide an objective measure of the vascular effects of any therapeutic intervention aimed at risk factor modification. Ultimately, our future health will depend on carefully balancing the benefits of early diagnosis and treatment in high-risk youth with the long-term risk of CVD. The application of SVMs in the pediatric population will help us achieve this balance.

Infection of frog neurons with vaccinia virus permits in vivo expression of foreign proteins.

Vaccinia virus can be used to infect cells in the CNS of frogs, Xenopus laevis, and Rana pipiens, both in vivo and in vitro. In vivo infections were accomplished by injection of viral solution into the tectal ventricle of stage 40-48 tadpoles or by local injections into distinct neural regions. Infections with high titer of virus injected into the ventricle resulted in the majority of cells in the brain expressing foreign protein, while cells in the retina and optic nerve showed no expression. Infection with lower viral titers resulted in fewer infected cells that were distributed throughout the otherwise normal tissue. Intense expression of foreign protein in the brain was observed 36 hr after injection and remained high for at least 4 days. Infected animals developed normally and had the same number of cells in the optic tectum as control animals. Infection with a recombinant virus carrying the gene for Green Fluorescent Protein labels neurons, so that infected cells can be observed in vivo. Vaccinia virus provides a versatile means to alter proteins in distinct populations of neurons in amphibia.

Spaced stimuli stabilize MAPK pathway activation and its effects on dendritic morphology.

Memory storage in mammalian neurons probably depends on both biochemical events and morphological alterations in dendrites. Here we report an activity-dependent stabilization of the MAP kinase (MAPK) pathway, prominent in hippocampal dendrites. The longevity of the signal in these dendrites was increased to hours when multiple spaced stimuli were used. Likewise, spaced stimuli and MAPK activation were critical for protrusion of new dendritic filopodia that also remained stable for hours. Our experiments define a new role for stimulus-specific responses of MAPK signaling in activity-dependent neuronal plasticity. The local biochemical signaling in dendrites complements MAPK signaling in gene expression. Together, these processes may support long-lasting behavioral changes.

The scaffold protein, Homer1b/c, regulates axon pathfinding in the central nervous system in vivo.

Homer proteins are a family of multidomain cytosolic proteins that have been postulated to serve as scaffold proteins that affect responses to extracellular signals by regulating protein-protein interactions. We tested whether Homer proteins are involved in axon pathfinding in vivo, by expressing both wild-type and mutant isoforms of Homer in Xenopus optic tectal neurons. Time-lapse imaging demonstrated that interfering with the ability of endogenous Homer to form protein-protein interactions resulted in axon pathfinding errors at stereotypical choice points. These data demonstrate a function for scaffold proteins such as Homer in axon guidance. Homer may facilitate signal transduction from cell-surface receptors to intracellular proteins that govern the establishment of axon trajectories.

Intracellular cleavage of hepatitis C virus RNA and inhibition of viral protein translation by hammerhead ribozymes.

To determine the effects of hammerhead ribozymes against hepatitis C virus (HCV) RNA on viral protein translation, a luciferase reporter gene vector, pCMV/T7-NCRCdelta-luc, was constructed containing the 5'-noncoding region (5'-NCR) and part of the core region of HCV. Four ribozymes, Rz1-Rz4, were designed to cleave at nucleotide positions 136-160, 313-337, 496-520, and 373-388, respectively. Each ribozyme cleaved the target RNA at expected positions under cell-free conditions. Rz2 and Rz4 significantly suppressed translation of NCRCdelta-luc RNA by 71 and 49%, respectively. Translation of control luciferase mRNA lacking viral elements was not affected by the ribozymes. Furthermore, when NCRCdelta-luc RNA and ribozymes were cotransfected into cells, Rz2 and Rz4 significantly suppressed expression by 73 and 56%, respectively. In contrast, cleavage-deficient ribozymes with a point mutation in the hammerhead domain had no significant effect. To determine the effects of endogenously produced ribozymes, eukaryotic expression vectors for Rz2 and Rz4 were constructed. Cotransfection of the vectors with CMV/T7-NCRCdelta-luc showed suppression of luciferase activities to 50 and 61%, respectively. Moreover, transfection of pCMV/T7-NCRCdelta-luc into stable Rz2 and Rz4 producer cells also showed substantial inhibition of luciferase activity. Ribozymes directed against the HCV genome can substantially and specifically inhibit viral gene expression under intracellular conditions.