A continuous fluorescence displacement assay for the measurement of phospholipase A2 and other lipases that release long-chain fatty acids.

1. A new continuous fluorescence assay for phospholipase A2 is described which involves the displacement of the highly fluorescent fatty-acid probe 11-(dansylamino)undecanoic acid from rat liver fatty-acid-binding protein by long-chain fatty acids released as a result of phospholipase A2-catalysed hydrolysis of phospholipids. The initial rate of decrease in fluorescence is linearly related to enzyme activity. 2. The assay will detect enzyme activity down to about 10 pmol/min per ml and gives a linear response up to about 10 nmol/min per ml. 3. The assay will work with all phospholipids that have been tested including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol. Substrates carrying a net negative charge showed the highest rates of hydrolysis. 4. The assay will work, in principle, with an enzyme catalysing the release of long-chain fatty acids from a fatty-acylated substrate. This has been confirmed with pancreatic lipase and cholesterol esterase.     

Phorbol ester-induced differentiation permits productive human cytomegalovirus infection in a monocytic cell line

The susceptibility of four different human cell lines (HUT 102, THP-1, MOLT-4, and HL-60) to infection by human CMV (HCMV) was studied. Only HUT 102 was susceptible and only immediate-early gene products were produced. However, THP-1, a monocytic cell line, could be infected by HCMV with a full cycle of replication after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which produced differentiation of the cell line into cells with characteristics of mature macrophages. Late (structural) Ag were demonstrated, as were infectious virions as detected by electron microscopy and infectious center assay. HL-60, a promyelocytic cell line, was not susceptible to HCMV infection after treatment with TPA despite differentiation into adherent cells with properties of macrophages, suggesting that cellular lineage was important. Treatment with TPA after infection resulted in a greatly reduced frequency of infected cells, suggesting that pretreatment was essential. Furthermore, continued presence of TPA was unnecessary after differentiation was induced. This study establishes the precedent of productive HCMV infection in human monocytic cells. The potential mechanism and relevance of enhanced replication induced by TPA are discussed.     

Stability of Suspensions of Influenza Virus Dried to Different Contents of Residual Moisture by Sublimation In Vacuo

After freezing, suspensions of influenza virus were dried by sublimation of water in vacuo to contents of residual moisture of 3.2, 2.1, 1.7, 1, or 0.4%. The stability of the several suspensions was determined by an accelerated storage test. Based on the times predicted for the dried preparations stored at different temperatures to lose 1 log of infectivity titer, the order of stability in relation to residual moistures was as follows: 1.7% > 2.1% > 1% > 3.2% > 0.4%.     

An apparent lack of stereospecificity in the reaction catalysed by deoxyribose 5-phosphate aldolase due to methyl-group rotation and enolization before product release.

In the reaction catalysed by deoxyribose 5-phosphate aldolase (2-deoxy-D-ribose 5-phosphate acetaldehyde-lyase, EC 4.1.2.4) from Salmonella typhimurium, almost complete equilibration of the methyl-group protons of the product, acetaldehyde, occurs before its release from the enzyme surface. This phenomenon does not allow the stereo-chemical course of the reaction to be determined by using hydrogen-isotope labelling of the methyl group to generate a chiral centre.     

Physical Anthropology and the dental and medical specialties

The rise of two sub-specialties in Physical Anthropology traces back to the Anatomy Departments of Schools of Medicine in Germany and France during the nineteenth century. The study of human diversity in bones and bodies was largely by medically-trained anatomists. There developed Medical Anthropology and Dental Anthropology, employing osteometry and craniometry on the skeleton, somatometry and cephalometry on the living body. As a result cross-sectional studies gave way to longitudinal studies and X-ray techniques were added to purely mensurational procedures. In Medical Anthropology the specialties most directly concerned are pediatrics, plastic surgery, endocrinology, and orthopaedics. In Dental Anthropology the specialties most directly concerned are pedodontics, orthodontics, oral surgery, and prosthodontics. The contributions of Physical Anthropology to each is discussed.     

Soybean lecithin-based extender as an alternative for goat sperm cryopreservation

The aim of this study was to evaluate the effect of different concentrations of soybean lecithin (SL) in extenders for sperm goat cryopreservation. Sexually mature male Saanen goats (n = 4) were used, and the ejaculates were obtained using an artificial vagina method. The semen samples were pooled and diluted in a skim milk-based extender (control group; CG) or Tris extender supplemented with SL at different concentrations (G1 = 0.04%, SL G2 = 0.08% SL and G3 = 0.16%) for a final concentration of 240 × 10⁶ spermatozoa/mL. The semen samples were packed in straws (0.25 mL), frozen using an automated system and stored in liquid nitrogen (?196 °C). After thawing (37 °C/30 s), the samples were evaluated for sperm quality parameters, including sperm motility, membrane integrity, acrosome integrity and mitochondrial activity. No significant difference was observed among the experimental and control groups for all of the parameters (P > 0.05). However, even though the control group presented a significantly lower mitochondrial membrane potential compared to fresh semen (P < 0.05), the same did not occur for the extender supplemented with soybean lecithin, that is, it did not differ from fresh sperm (P > 0.05). The extender containing soybean lecithin at different concentrations preserved the sperm quality parameters in a manner similar to the conventional skim milk-based extender. Thus, it is concluded that an extender containing soybean lecithin as the lipoprotein source can be used for freezing goat semen.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Association of Pro-Inflammatory Cytokines and Iron Regulatory Protein 2 (IRP2) with Leishmania Burden in Canine Visceral Leishmaniasis

Leishmania infantum infection in humans and dogs can evolve with a wide range of clinical presentations, varying from asymptomatic infections to visceral leishmaniasis. We hypothesized that the immune response elicited by L. infantum infection could modulate whether the host will remain asymptomatic or progress to disease. A total of 44 dogs naturally infected with L. infantum were studied. Leishmania burden was estimated in the blood and spleen by qPCR. The expression of IFN-γ, TNF-α, IL-10 and Iron Regulatory Protein 2 (IRP2) were determined in the spleen by quantitative PCR. Sera cytokines were evaluated by ELISA. Dogs were grouped in quartiles according parasite burden. Increased expression of IFN-γ and TNF-α was associated with reduced Leishmania burden, whereas increased IL-10 and IRP2 expressions were associated with higher Leishmania load. Increased plasma albumin and IFN-γ expression explained 22.8% of the decrease in parasite burden in the spleen. These data confirm that lower IFN-γ response and higher IL-10 correlated with increased parasite load and severity of the visceral leishmaniasis in dogs. The balance between the branches of immune response and the intracellular iron availability could determine, in part, the course of Leishmania infection.     

The chemical modification of cysteine-69 of rat liver fatty acid-binding protein (FABP): a fluorescence approach to FABP structure and function

Summary Hepatic-FABP was labelled at cysteine-69 with the fluorescent environmentally sensitive reporter group AEDANS. The labelled protein had an emission maximum at 465 nm indicating that cysteine-69 was buried in a non-polar environment. The modified protein was still able to bind ligands such as oleic acid, oleoyl CoA and haem. The affinity of AEDANS-FABP for haem was unaltered as compared with the native protein indicating that cysteine-69 must be remote from the ligand binding site. The binding of oleic acid did not significantly perturb the fluorescence emission spectrum of the fluorescent reporter group suggesting that there are not large conformational changes in the region of cysteine-69 on fatty acid binding. The binding of stoichiometric amounts of oleoyl CoA was accompanied by a small fluorescence enhancement which suggests that fatty acyl CoAs may interact with other regions of the FABP molecule not involved in fatty acid binding.Abbreviations FABP Fatty Acid-Binding Protein - AEDANS 5-[2-(Acetyl)aminoEthyl]Aminonaphthalene-1-Sulphonic Acid - AAEDANS 5-[2-(Iodoacetyl-AminolEthyl] aminonaphthalene-1-Sulphonic Acid - DTNB 5,5Dithiobis-(2-Nitrobenzoic acid)