Isolation and characterization of propagable cell lines (HUNC) from the androgen-sensitive Dunning R3327H rat prostatic adenocarcinoma

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC- E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.      

Zinc replenishment reduces esophageal cell proliferation and N- nitrosomethylbenzylamine (NMBA)-induced esophageal tumor incidence in zinc-deficient rats

Previous work has shown that sustained increased and decreased cell proliferation, induced by dietary zinc deficiency and caloric restriction respectively, influence the course of N- nitrosomethylbenzylamine (NMBA)-induced esophageal carcinogenesis in rats. The present study considered whether the increased cell proliferation and esophageal tumor incidence induced by zinc deficiency are reversed upon zinc replenishment. Weanling rats were maintained initially on a deficient diet containing 4 p.p.m. zinc. After 5 weeks, carcinogen-treated animals were given six intragastric doses of NMBA (2 mg/kg twice weekly). Controls were untreated. After the second NMBA dose, the rats were divided into three dietary groups. One group was continued on the deficient diet, while the other two groups were switched to diets containing either 75 or 200 p.p.m. zinc, with half of the members in each group fed ad libitum and half pair-fed with deficient rats. NMBA-untreated controls were similarly replenished. At various time points, esophageal cell proliferation was assessed in five animals from each group by immunohistochemical detection of cells in S phase, with in vivo 5-bromo-2'deoxyuridine labeling. At 11 weeks after the first dose, esophageal tumor incidence was greatly reduced, from 100% in the deficient group to 26 and 14% respectively in the replenished groups fed ad libitum 75 and 200 p.p.m. zinc and to 14 and 11% respectively in the replenished groups pair-fed 75 and 200 p.p.m. zinc. In addition, the number of tumors per esophagus was reduced from 9.93 +/- 4.25 in deficient rats, to a range of 0.11 +/- 0.31-0.30 +/- 0.54 in replenished animals. Following zinc replenishment, esophageal cell proliferation, as measured by labeling index (LI), the number of labeled cells and the total number of cells, was markedly decreased in NMBA-untreated and -treated esophagi as compared with those in corresponding deficient esophagi. Thus, the esophageal cell proliferation induced by zinc deficiency is reversed by zinc replenishment and replenished animals have a markedly lower incidence of esophageal tumors.      

Symptom dimensions in recent-onset schizophrenia and mania: a principal components analysis of the 24-item Brief Psychiatric Rating Scale

Previous four- and five-factor solutions of the 18-item Brief Psychiatric Rating Scale (BPRS) suggested the possibility of an affective dimension in psychosis. A principal components analysis was used to analyze psychiatric symptom data rated on an expanded 24-item version of the BPRS. BPRS data were collected during a period of acute psychotic and affective illness with 114 young adult, recent-onset schizophrenia and schizoaffective patients and 27 bipolar manic patients. Principal components analyses of the 18-item and 24-item BPRS indicated a four-factor solution was the most interpretable. Principal components analysis of the 24-item BPRS produced a clear mania factor characterized by high loadings from items added to the 18-item BPRS, which included elevated mood, motor hyperactivity, and distractibility. This factor solution suggests that the 24-item BPRS allows for an expanded assessment of affective symptoms relating to a manic dimension. Potentially important symptoms that were added to the traditional 18-item version, namely suicidality, bizarre behavior, and self-neglect, also make clear contributions to other factors.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase expression in rat brain during development

This study reports the expression of the ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene (PFKFB3) (PFK-2/FBPase-2) in different stages of rat brain development. Northern blot and RT-PCR analysis demonstrated that ubiquitous PFK-2/FBPase-2 is expressed in rat brain from embryonic to adult life and shows a transient increase 1 day before birth, coincident with the maximum concentration of Fru-2,6-P(2) and PFK-2 activity. The levels of brain PFK-2/FBPase-2 gene expression as well as the enzymatic activity and the concentration of Fru-2,6-P(2) appear to be remarkably constant during adult life, without significant differences in the brain hippocampus, cortex, cerebellum or striatum areas.     

A biochemical and pharmacological examination of Rhamphiophis oxyrhynchus (Rufous beaked snake) venom.

The venom of R. oxyrhynchus, a member of the psammophiine subfamily of the colubrid assemblage, was examined for biological activity using biochemical and pharmacological techniques. Venom displayed a high protein content, a complex electrophorectic profile and PLA2 activity but no detectable proteolytic or haematological activities. In the chick biventer cervicis nerve muscle preparation, venom (1-10 microg/ml) displayed postsynaptic neurotoxic activity as evidenced by inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches and responses to exogenous acetylcholine (1 mM) and carbachol (20 microM). This inhibitory effect was poorly reversible by washing. Venom (30-50 microg/ml) caused a rapid and readily reversible inhibition of direct (0.1 Hz, 2 ms, supramaximal V) twitches of the chick biventer cervicis nerve muscle preparation without morphological changes to the muscle fibers. Venom (30-100 microg/ml) inhibited electrically-evoked (0.2 Hz, 0.3 ms, 70-100 V) twitches of the prostatic segment of the rat vas deferens. This inhibitory effect was not significantly attenuated by 8-phenyltheophylline (8-PT; 20 microM), idazoxan (1 microM), a combination of ranitidine (0.2 microM) and thioperamide (10 microM) or capsazepine (10 microM). Venom (5 mg/kg) induced hypotension with subsequent cardiovascular collapse in the anaesthetised rat. The cardiovascular collapse was prevented by artificial respiration of the animals prior to venom administration. The biological activities demonstrated by R. oxyrhynchus venom may aid in prey envenomation strategies such as prey immobilisation. This study provides further evidence that colubrid venoms are comprised of multiple components which can display a variety of actions, some of which may be novel, therefore reinforcing the largely untapped potential of colubrid venoms.     

Expansion of Patient Education Programming Regarding Live Donor Liver Transplantation via Virtual Group Encounters During the COVID-19 Pandemic

During the coronavirus 2019 pandemic we converted our liver transplant waitlist candidate education and support program to a virtual format and expanded it to include ongoing engagement sessions aimed to educate and empower patients to maximize opportunity for live donor liver transplantation. Over a period of 6 months from April 2020 to Sept 2020 we included 21 patients in this pilot quality improvement program. We collected data regarding patient response and potential donor referral activity. Overall, patient response was positive, and some patients saw progress toward live donor liver transplantation by fostering inquiry of potential live liver donors. Optimization of logistical aspects of the program including program flow, technology access, and utilization is required to enhance patient experience. Long-term follow-up is needed to assess impact on the outcome of transplantation rates. Future data collection and analysis should focus on assessment of any potential disparity that may result from utilization of virtual programming. Herein we provide a framework for this type of virtual program and describe our experience.