Stimulation of polypeptide synthesis by spermidine at the level of initiation in rabbit reticulocyte and wheat germ cell-free systems

It is shown that the stimulation of eukaryotic polypeptide synthesis by spermidine is due to the stimulation at the level of initiation by following reasons. The incorporation of formylmethionine into polypeptides was stimulated by spermidine at the same degree to the incorporation of leucine into polypeptides. Fluorography of the polypeptides formed showed that the number of chains of individual protein synthesized was larger when spermidine was added. The formation of the complex of Met-tRNA_f, globin mRNA and 40-S ribosomal subunits was stimulated by spermidine.     

Kinetic mechanism of the Cu(II) enzyme galactose oxidase

The steady-state kinetics of four redox reactions catalyzed by galactose oxidase have been determined. The alcohol substrate used in each case was galactose; the four oxidant substrates used were O₂, IrCl₆^2?, porphyrexide, and Fe(CN)₆^3?. With the exception of the last reagent, saturation behavior is exhibited by all substrates. Double reciprocal plots of rate data obtained varying one substrate at various concentrations of the other are intersecting for all pairs that exhibited saturation behavior. Thus, these reactions are kinetically sequential processes involving single central complexes. These complexes involve enzyme, galactose, and one molecule of oxidant, whether or not the oxidant is a one- or two-electron acceptor. This result indicates that for one-electron oxidants, an enzyme-alcohol-derived radical species may exist as a transient prior to the reaction of the second electron equivalent of oxidant. A similar substrate

transient is postulated in the reaction involving O₂. The inhibition by H₂O₂ has also been studied in detail. H₂O₂ apparently binds to the enzyme at two sites. The nature of alcohol and O₂ binding to the enzyme Cu(II) is discussed in light of these kinetic results.     

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)¹. While improvements in several gene delivery methods have been described^2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor¹¹, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.     

Further studies on the effect of aldosterone on electrical resistance of toad bladder

The fall in transepithelial electrical resistance which accompanies aldosterone stimulation of short-circuit current ($\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$) in toad urinary bladder has been studied further to evaluate the possible causal role of this response in hormonal stimulation of Na⁺ transport. A steady-state change in tissue conductance was found to depend upon both the simultaneous stimulation of transport by the steroid and the metabolic state of the tissue. Changes in metabolic state alone did not alter resistance. A sustained increase in Na⁺ transport, dependent on pretreatment with aldosterone and elicited by addition of glucose, could be obtained without a sustained decrease in resistance. Amiloride, an inhibitor of Na⁺ uptake, produced changes in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ that were linearly correlated with its effects on tissue conductance. On the basis of the $\text{conductance}\text{-I}{}_{\mathrm{<mtext>sc</mtext>}}$ relationship with amiloride, the $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ response to aldosterone was about two-fold higher than would be predicted from its effects on conductance alone. Despite the apparent lack of a simple quantitative dependence of the change in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ on the change in conductance when the response is fully developed, the results suggest that conductance changes may mediate the initial or early stage of the response.     

The stimulus-secretion coupling of glucose-induced insulin release: XVI. A glucose-like and calcium-independent effect of cyclic AMP

Theophylline increases the synthesis of proinsulin and, to a lesser extent, that of non insulinic peptides in isolated islets of Langerhans. Similar to its stimulant action on ⁴⁵Ca²⁺ uptake by insular tissue, the preferential stimulant action of theophylline on proinsulin biosynthesis is most marked at low glucose concentration (4.2 mM). It apparently represents a Ca²⁺-independent process. Since theophylline does not augment glucose uptake by the isolated islets, the glucose-like enhancing action of theophylline on both ⁴⁵Ca²⁺ uptake and proinsulin synthesis could be due, in part at least, to a cyclic AMP-mediated stimulation of glycogenolysis.     

The crystal structure of uncomplexed-hydrated cyclooctaamylose

Cyclooctaamylose crystallizes from aqueous solution with space-group symmetry P2₁ and lattice parameters: $\text{a}\text{= 20.253(8),}\text{b}\text{= 10.494(5),}\text{c}\text{= 16.892(6)}$ A and β = 105.32(1)^o, Z = 2; the apparent formular per asymmetric unit is C₄₈H₈₀O₄₀·17H₂O. The macrocycle is in an open conformation but displays significant deviations from ideal eight fold molecular symmetry. Of the 19 water molecules thus far located, four of which have occupancy factors of one half, 12 may be characterized as being in the torus of the cycloamylose.     

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules ¹. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes ² and restrict their environment to a more favorable low oxygen pressure ³. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria ^4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor ⁶. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)^7,8 has also been associated with severe disease ⁹.One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps ¹⁰. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM ¹¹. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian ¹² and Mozambican patients ¹³, (although not in Malian ¹⁴).With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay ¹⁵. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.     

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis¹. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain², a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)^3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity⁵.A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method⁶ to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum⁷ as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.CP/CPPS is primarily characterized by the presence of tactile allodynia⁴. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain^8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.