Location of the isoleucine arrest point in CHO and 3T3 cells

An isoleucine arrest point in G1 was determined by two methods for CHO and 3T3 cells. In the first method the fraction of cells entering S after isoleucine deprivation was assessed by [³H]thymidine labelling and autoradiography. In the second method cells entering S after isoleucine deprivation were identified by double-label autoradiography using [³H] and [¹⁴C]thymidine. From the fraction of cells entering S, determined by the two methods, the arrest point in G1 (and entry into G0) is located within the last 40 min of G1.     

Reduction in size of prolactin-secreting tumours in men treated with pergolide.

The effect of pergolide mesylate was studied in two previously untreated men with large prolactinomas and exceptionally high prolactin concentrations. The study was designed to determine whether pergolide would be effective in alleviating symptoms, correcting hormonal abnormalities and shrinking the tumour. Starting with 50 micrograms daily the dose of pergolide was slowly increased over 10 weeks to 1 mg once daily, when repeat assessment was performed. Both patients reported complete relief of symptoms, with no side effects. Serum prolactin concentration was suppressed to normal in both subjects, and evidence to suggest tumour shrinkage was observed. Pergolide appears to be effective treatment for men with large prolactinomas.     

A Mathematical Framework for Combining Decisions of Multiple Experts toward Accurate and Remote Diagnosis of Malaria Using Tele-Microscopy

We propose a methodology for digitally fusing diagnostic decisions made by multiple medical experts in order to improve accuracy of diagnosis. Toward this goal, we report an experimental study involving nine experts, where each one was given more than 8,000 digital microscopic images of individual human red blood cells and asked to identify malaria infected cells. The results of this experiment reveal that even highly trained medical experts are not always self-consistent in their diagnostic decisions and that there exists a fair level of disagreement among experts, even for binary decisions (i.e., infected vs. uninfected). To tackle this general medical diagnosis problem, we propose a probabilistic algorithm to fuse the decisions made by trained medical experts to robustly achieve higher levels of accuracy when compared to individual experts making such decisions. By modelling the decisions of experts as a three component mixture model and solving for the underlying parameters using the Expectation Maximisation algorithm, we demonstrate the efficacy of our approach which significantly improves the overall diagnostic accuracy of malaria infected cells. Additionally, we present a mathematical framework for performing ‘slide-level’ diagnosis by using individual ‘cell-level’ diagnosis data, shedding more light on the statistical rules that should govern the routine practice in examination of e.g., thin blood smear samples. This framework could be generalized for various other tele-pathology needs, and can be used by trained experts within an efficient tele-medicine platform.     

Human plasma platelet-activating factor acetylhydrolase. Purification and properties

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid synthesized by a variety of cell types upon appropriate stimulation. PAF is a potent hypotensive factor and it activates platelets and inflammatory cells at concentrations as low as 10(-10) M. Removal of the acetyl moiety at the sn-2 position abolishes the biological activity and this reaction is catalyzed by a specific acetylhydrolase present in plasma and animal tissues. Ultracentrifugation in density gradients showed that 30% of the activity is associated with high density lipoproteins and 70% with low density lipoproteins. We have purified the plasma low density lipoprotein-associated activity to near homogeneity using a rapid assay based on the separation of [3H]acetate from 1-O-alkyl-2-[3H]acetyl-sn-glycerol-3-phosphocholine on disposable reversed-phase columns. The enzyme was purified by 25,000-fold and approximately 10% of the starting activity was recovered. Plasma PAF-acetylhydrolase has an apparent molecular weight of 43,000, does not require calcium, has preference for micellar versus monomeric substrate, and exhibits surface dilution kinetics. The purified protein has an apparent Km of 13.7 microM and a Vmax of 568 mumol/h/mg with micellar PAF. It can act both on 1-O-alkyl and 1-acyl substrates and on ethanolamine analogs of PAF. However, the enzyme has a marked preference for the sn-2 acetyl residue and therefore can be considered as a specific PAF-acetylhydrolase.     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

A single mutation in 16S rRNA that affects mRNA binding and translation-termination.

A single base change in 16S rRNA (C726 to G) has previously been shown to have a dramatic effect on protein synthesis in E. coli (1). This paper more specifically details the effects of the mutation on mRNA binding and translation-termination. The in vitro technique of toeprinting (2) was used to demonstrate that 30S subunits containing the mutation 726G had an altered binding affinity for mRNA by comparison to the wild type. In addition, expression of the mutant ribosomes in vivo resulted in exclusive suppression of the UGA nonsense codon. This effect was supported by in vitro studies that showed the mutant ribosomes to have an altered binding affinity for Release Factor-2.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.