Selective CREB-dependent cyclin expression mediated by the PI3K and MAPK pathways supports glioma cell proliferation.

The cyclic-AMP response element binding (CREB) protein has been shown to have a pivotal role in cell survival and cell proliferation. Transgenic rodent models have revealed a role for CREB in higher-order brain functions, such as memory and drug addiction behaviors. CREB overexpression in transgenic animals imparts oncogenic properties on cells in various tissues, and aberrant CREB expression is associated with tumours. It is the central position of CREB, downstream from key developmental and growth signalling pathways, which gives CREB this ability to influence a spectrum of cellular activities, such as cell survival, growth and differentiation, in both normal and cancer cells. We show that CREB is highly expressed and constitutively activated in patient glioma tissue and that this activation closely correlates with tumour grade. The mechanism by which CREB regulates glioblastoma (GBM) tumour cell proliferation involves activities downstream from both the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways that then modulate the expression of three key cell cycle factors, cyclin B, D and proliferating cell nuclear antigen (PCNA). Cyclin D1 is highly CREB-dependent, whereas cyclin B1 and PCNA are co-regulated by both CREB-dependent and -independent mechanisms. The precise regulatory network involved appears to differ depending on the tumour-suppressor phosphatase and tensin homolog status of the GBM cells, which in turn allows CREB to regulate the activity of the PI3K itself. Given that CREB sits at the hub of key cancer cell signalling pathways, understanding the role of glioma-specific CREB function may lead to improved novel combinatorial anti-tumour therapies, which can complement existing PI3K-specific drugs undergoing early phase clinical trials.

Functional incompatibility between the fertilization systems of two allopatric populations of Callosobruchus maculatus (Coleoptera: Bruchidae).

Recent studies indicate that postcopulatory sexual selection may represent an important component of the speciation process by initiating reproductive isolation via the evolutionary divergence of fertilization systems. Using two geographically isolated populations of the polyandrous beetle Callosobruchus maculatus, we investigated divergence in fertilization systems by determining the extent of postcopulatory functional incompatibility. Through reciprocal, cross-population matings we were able to separately estimate the effects of male and female population origin and their interaction on the extent of last-male sperm precedence, female receptivity to further copulation and female oviposition. Our results indicate partial incompatibility between the fertilization systems of the two populations at all three functional levels. Males derived from the same population as females outcompete rival, allopatric males with respect to sperm preemption, sperm protection, and ability to stimulate female oviposition. This pattern is reciprocated in both populations indicating that postcopulatory, prezygotic events represent important mechanisms by which between-population gene flow is reduced. We suggest the partial gametic isolation observed is a by-product of the coevolution of male and female fertilization systems by a process of cryptic female choice. Our results are consistent with a mechanism akin to conventional mate choice models although they do not allow us to reject antagonistic sexual coevolution as the mechanism of cryptic female choice.

The impact of renal dysfunction on aprotinin pharmacokinetics during cardiopulmonary bypass.

UNLABELLED: Aprotinin is a serine protease inhibitor that undergoes metabolism in the kidney. Because elimination is almost entirely renal, the clearance of aprotinin may be reduced in patients with renal insufficiency. Unfortunately, there are no data regarding aprotinin pharmacokinetics in cardiac surgical patients with renal insufficiency or end-stage renal disease (ESRD) undergoing cardiopulmonary bypass (CPB). We, therefore, determined the clearance (ApCl) and elimination half-life (T1/2) of aprotinin in 26 cardiac surgical patients with normal and abnormal renal function (creatinine clearance [CrCl] 0-122 mL/min) undergoing CPB. Subjects were given a 2 million kallikrein inhibiting unit (KIU) initial dose of aprotinin, followed by a 0.25 million KIU/h infusion. No aprotinin was added to the pump prime. Plasma aprotinin concentrations were sampled at 30 min after completion of the loading dose, 30 and 60 min after the onset of CPB, at the end of CPB, and at 8, 24, and 32 h after completion of the loading dose. ApCl was directly related and the elimination T1/2 inversely related to CrCl (r = 0.75 and 0.42, respectively). In patients with a CrCl >50 mL/min, the T1/2 and ApCl were 7.8 h and 53 mL/min, respectively, compared with 19.9 h and 25 mL/min (P < 0.05, P < 0.002, respectively) for patients with ESRD. In conclusion, ApCl is reduced, and T1/2 is prolonged in patients with renal insufficiency or ESRD undergoing CPB. Dosing modifications may be necessary for patients with abnormal renal function undergoing cardiac surgery. IMPLICATIONS: Because aprotinin is metabolized and eliminated in the kidney, its clearance may be reduced in patients with renal insufficiency. Our data suggest that aprotinin clearance is reduced, and aprotinin half-lives are prolonged in patients with renal insufficiency undergoing CPB. Dosing modification may therefore be indicated when aprotinin is administered to these patients for cardiac surgery.

Task Group for Orthodontics Report.

The UK Specialist Review Group of the General Dental Council's Education Committee has been charged with taking forward the recommendations in the Chief Dental Officer's report 'UK Specialist Dental Training'. The Specialist Review Group has, in turn, established a number of specialty task groups. This report is from the Task Group for Orthodontics. It was submitted in May 1996.

Potentiometric enzyme channeling immunosensor for proteins.

A potentiometric immunosensor for the detection of human IgG has been developed using an asymmetric, ion-selective membrane with immobilized adenosine deaminase and IgG. A protein A-alkaline phosphatase conjugate binds to the immobilized IgG, creating a bienzymatic catalytic layer. In the presence of sample IgG, the conjugate does not bind to the membrane. Instead, the intermediate in the two-step reaction (adenosine) must diffuse to the membrane surface, reducing the rate of product (ammonium) formation within the diffusion layer detected by the membrane. The immunosensor demonstrated is for the determination of IgG. A simplified model is described to predict the maximum rate enhancement for the 'channeled' versus 'unchanneled' reaction mechanisms.

Steroidogenesis and testosterone metabolism in cultured principal cells from the ram epididymis.

To determine if ram principal cells can synthesize or metabolize testosterone, or metabolize other steroids present in rete testis fluid, principal cells from the initial segment, central caput, and proximal corpus epididymidis were isolated and cultured in a floating collagen matrix with medium containing 20% dialyzed rete testis fluid. In the first experiment, each matrix was washed twice in testosterone-free medium on day 2.8, transferred into culture medium containing 100 nM of a tritiated steroid and incubated for 4 hours at 34 C. The tritiated steroids were pregnenolone, 5-androstene-3 beta,17 beta-diol, progesterone, 4-androstene-3,17-dione, testosterone, and dihydrotestosterone. Since testosterone was not formed from 5-androstene-3 beta,17 beta-diol or 4-androstene-3,17-dione, testosterone synthesis by ram principal cells is unlikely Pregnenolone and 5-androstene-3 beta,17 beta-diol were not metabolized and only slight metabolism of dihydrotestosterone occurred. Progesterone, 4-androstene-3,17-dione, and testosterone were metabolized to 5 alpha-reduced products tentatively identified as 5 alpha-pregnane-3,20-dione and 5 alpha-pregnan-3 beta-ol-20-one and/or 5 alpha-pregnan-20 alpha-ol-3-one; 5 alpha-androstane-3,17-dione and 5 alpha-androstan-3 alpha-ol-17-one, and dihydrotestosterone, respectively. The second experiment evaluated testosterone metabolism by both cultured principal cells and minced epididymal tissue. On day 1 of culture, during 12 hours the accumulation of dihydrotestosterone in medium from cells of the central caput was 48 X and 1.1 X that in medium from cells of the initial segment and proximal corpus epididymidis.(ABSTRACT TRUNCATED AT 250 WORDS)

Culture of principal cells from the ram epididymis. A comparison of the morphology of principal cells in culture and in situ.

A procedure was developed to isolate and culture principal cells from the initial segment, central caput, distal caput, and proximal corpus epididymidis. The morphology of cultured cells and of cells in situ was compared. Over four to ten days, principal cells cultured in a floating collagen matrix with serum-free medium formed clusters that developed into either large sheets of columnar cells or tubular structures of cuboidal cells. Structural polarity was evident and junctional complexes reformed. The distribution and relative abundance of organelles in principal cells in situ differed depending on the region examined, and most of these regional characteristics were retained by principal cells in culture. Microvilli and membrane-bound vesicles were less conspicuous in cultured cells. Cultured principal cells were shorter than principal cells in situ, but were of similar volume. The high purity (90%) and viability (70%) of principal cells after seven to ten days in culture, their retention of morphologic characteristics unique to the region of origin, and the formation of function units are evidence that such cultures should be valuable for studying regional differences in the function of principal cells.

Inhibition of testosterone metabolism in cultured rat epididymal principal cells by dihydrotestosterone and progesterone.

To evaluate the effects of steroids entering the epididymis in rete testis fluid on testosterone (T) metabolism by the epididymal epithelium, principal cells were isolated from the proximal caput, distal caput or corpus epididymidis by enzymatic dissociation and elutriation and were cultured at 34 degrees C within a floating collagen matrix. The culture medium was supplemented with T, dihydrotestosterone (DHT), T plus estradiol-17 beta (T + E) or T plus progesterone (T + P) at concentrations which were approximately physiologic. Metabolism of T by principal cells incubated for 2.5 days with DHT was lower (P less than 0.05) than for control cells cultured with T. Inclusion of E or P in the culture medium lowered (P less than 0.05) metabolism of T by principal cells from each region. However, principal cells cultured with T + P for 2.5 days and then washed and cultured for 12 h with T alone, metabolized T as well (P less than 0.05) as cells never exposed to P. In marked contrast to the persistent suppressive effect of DHT, the suppressive effect of P on metabolism of T is rapid, direct and rapidly reversible. Thus, metabolism of T by principal cells in the epididymal epithelium may be modulated by steroids (E + P) in rete testis fluid or by steroids (DHT) produced locally in the epididymis.

Influence of rete testis fluid on the metabolism of testosterone by cultured principal cells isolated from the proximal or distal caput of the rat epididymis.

Principle cells from 120 elutriations were used to improve procedures for culturing cells from the proximal or distal caput epididymidis. The criteria evaluated were metabolism of testosterone (T) to 5 alpha-reduced metabolites and cellular morphology after 6 days of culture. Isolated principal cells (greater than 90% viability) were cultured at 34 degrees C within a floating collagen matrix. Inclusion of transferrin or retinol in the culture medium increased the production of 5 alpha-reduced metabolites. Aggregation of principal cells before entrapment in the collagen matrix resulted in higher production of 5 alpha-reduced metabolites and more cells with a normal find structure than entrapment of dispersed cells in the matrix. Aggregated cells tended to form sheets or clusters, frequently arranged around a central lumen, with junctional complexes between adjacent cells. Cell polarity and morphologic features distinguishing principal cells from the proximal caput and distal caput epididymidis were retained. An average of 91% of the cells in aggregates were morphologically normal on Day 6 of culture in contrast to 5% for the single cells. Utilizing the improved culture procedure, we tested the hypothesis that ovine rete testis fluid (RTF) contains macromolecules which would aid in maintenance of a high rate of T metabolism. Principal cells were cultured in medium supplemented with 0 or 10% RTF, 10% ultrafiltrate of RTF (less than 10,000 daltons), or 10% newborn calf serum (NCS). Conversion of [3H]T to 5 alpha-reduced metabolites by cells from the proximal caput was twice that in cells from the distal caput on Day 6 of culture. Inclusion in the culture medium of 10% RTF or 10% NCS, but not 10% ultrafiltrate of RTF, increased (P less than 0.05) the production of 5 alpha-reduced metabolites by cells from both regions. We conclude that macromolecules in RTF or NCS are beneficial to maintenance of the ability to metabolize T by cultured principal cells, especially those from the proximal caput.

Influence of incubation in utero on motility and head-to-head agglutination of ejaculated rabbit spermatozoa.

Ejaculated spermatozoa were rendered immotile by incubation for 8 h at 37 degrees C in 2.9% sodium citrate. Immotile spermatozoa (5 x 10(6)) were surgically inseminated into the uterine lumen of does and samples were recovered from the uterus 5, 15, 30 and 60 min after insemination. Incubation in utero led to a resumption of progressive motility and induced head-to-head agglutination. Motility and head-to-head agglutination were highest (64 and 70% respectively) at 5 min, and declined (48 and 49%) by 60 min. The percentage of spermatozoa with an intact acrosome did not change during the in-utero incubation. When spermatozoa from a single ejaculate were evaluated in different females there was significant variation (P less than 0.01) in the reinitiation of motility and agglutination. Most agglutinated spermatozoa (greater than 96%) had an intact acrosomal membrane while acrosomal integrity of single spermatozoa differed greatly among females. We conclude that the agglutinated (motile with intact acrosomes) and non-agglutinated (usually immotile and with disrupted acrosomes) represent different populations within the uterine lumen.