Isolation and partial characterization of three isoamylases of Rhyzopertha dominica F. (Coleoptera: Bostrichidae)

Three isoamylases of Rhyzopertha dominica (termed RdA70, RdA79, and RdA90 according to their relative mobility in gel electrophoresis) were isolated by ammonium sulfate fractionation and hydrophobic interaction chromatography. RdA70 and RdA79 showed an optimal pH of 7.0, whereas for RdA90 the optimal pH was 6.5. The three isoamylases remained stable at 50 °C for 1 h, but at 60 °C, all lost 50% of their activity in 20 min and were completely inactivated in 1 h. RdA70 and RdA79 were inhibited by albumin extracts from wheat samples varying widely in amylase inhibitory activity; however, RdA90 was highly resistant to inhibition. β-Mercaptoethanol up to 30 mM increased the activity of the three isoamylases by 2.5-fold. The action pattern of the three isoamylases was typical of endoamylases; however, differences were observed on the hydrolytic efficiency rates measured as V_max/K_m ratio on starch, amylopectin, and amylose. The hydrolyzing action of RdA90 on starch and amylopectin (V_max/K_m = 90.4 ± 2.3 and 78.9 ± 6.6, respectively) was less efficient than that on amylose (V_max/K_m = 214 ± 23.2). RdA79 efficiently hydrolyzed both amylopectin and amylose (V_max/K_m = 260.6 ± 12.9 and 326.5 ± 9.4, respectively). RdA70 hydrolyzed starch and amylose at similar rates (V_max/K_m = 202.9 ± 5.5 and 215.9 ± 6.2, respectively), but amylopectin was a poor substrate (V_max/K_m = 124.2 ± 7.4). The overall results suggest that RdA70 and RdA79 appear to belong to a group of saccharifying isoamylases that breaks down long fragments of oligosaccharide chains produced by the hydrolytic action of RdA90. The simultaneous action of the three isoamylases on starch, aside from the high resistance of RdA90 to wheat amylase inhibitors, might allow R. dominica to feed and reproduce successfully on the wheat kernel.     

Development of Amblyospora campbelli (Microsporida: Amblyosporidae) in the mosquito Culiseta incidens (Thomson)

The complete life cycle of Amblyospora campbelli (Kellen and Wills, 1962) (Microsporida: Amblyosporidae) requires a two-host system involving the mosquito host, Culiseta incidens (Thomson), and an obligatory intermediate copepod host. The parasite has dimorphic spore development producing meiospores (haploid condition) and binucleated spores (diploid condition), either as an exclusive infection or simultaneously (within females only). This is the 1st known report of concurrent spore development within an adult mosquito host, and, therefore, shows the Amblyospora campbelli system to be uniquely different from other Amblyospora spp. cycles previously described. The significance of dimorphic spore development is discussed. In females, diplokaryotic meronts may invade oenocytes, causing a benign-type of infection. A blood-meal is required to initiate sporulation of the binucleate spore. The binucleate spore contains the sporoplasm involved in transovarial transmission. A 2nd sporulation sequence, primarily in adipose tissue, may involve both males and females. In this sequence, repeated merogonic division greatly increased the density of diplokaryotic meronts and generally involved most of the body of the host. Production of meiospores, unlike that for the binucleate spore, appeared to be spontaneous (i.e. no obligatory blood meal). Survivorship of male and female larval mosquitoes was nearly equal. Adult females spread the parasite in three ways: transovarial, transovum, and by meiospore deposition.     

Monoclonal antibodies against staphylococcal enterotoxins

MAb anti-Staphylococcal enterotoxins (MAb-SE) were produced in mice with a mixture of reference SE types A, B, C₁ and D at a concentration of 1 g each per mouse; the last booster injection was by intrasplenic route. Nine clones were chosen, two produced anti-SEB and anti-SED, one anti-SEA and anti-SEB, two anti-SED, two anti-SEB and one produced anti-SEC_1. The MAb-SE were partially purified as judged by PAGE–SDS. The partially purified antibodies could demonstrate the presence of SE in milk samples containing 0.5 g of toxin m l^–1.     

Calcium and body fat in peripubertal girls: cross-sectional and longitudinal observations

Objective: The objective was to investigate whether calcium intake is independently associated with body fat in peripubertal girls. Research Methods and Procedures: A total of 45 healthy premenarcheal girls (initially 10.5 ± 0.6 years of age) completed a 2‐year prospective observational study. Percent body fat and trunk fat (by DXA), height, weight, maturational stage, and eating attitudes (children's Eating Attitudes Test [EAT]) were measured at baseline and at 1 and 2 years. Physical activity (by questionnaire) and calcium intake (by calcium‐specific food frequency questionnaire and 3‐day food records) were assessed at 6‐month intervals. Results: Girls with 2‐year mean calcium intake below and above the median had similar age, height, lean mass, and maturational stage at baseline, but girls below the median had significantly higher baseline percentage body fat (29.3 ± 10.3% vs. 22.0 ± 6.8%, p < 0.01) and trunk fat (24.2 ± 10.6% vs. 15.8 ± 6.8%, p < 0.01). However, differences were no longer significant when covariates (most notably children's EAT dieting score) were considered. Regression analysis revealed that dieting score was a consistent positive predictor of percentage body and trunk fat at all cross‐sectional time‐points, accounting for >20% of the variance, but did not predict 2‐year change in percentage fat. Calcium intake did not enter longitudinal regression equations for 2‐year change in percentage fat. Discussion: In this group of girls, an inverse cross‐sectional association between calcium intake and body fat appeared to result from avoidance of foods high in calcium by girls who were concerned about their body weight or shape. Calcium intake was not associated with change in fat over time.     

A Simple Method for Mass-Production of Liposomes,in Particular Large Liposomes,Suitable for Delivery of Free Amino Acids to Filter Feeding Zooplankton

In marine fish larviculture the live feed organisms are often enriched in order to enhance their nutritional value. One of the challenges is to enhance the phospholipids (PL) content, and another is to enhance the content of specific water soluble nutrients, like free amino acids (FAA). There are a few studies where this has been achieved by the use of liposomes. The aim of this study was to develop a simple method for mass-production of liposomes within a size range of 1–5 μm a size range suitable to feed live food organisms. Furthermore, the liposomes should have a high FAA concentration and be stable under conditions typical for short-time enrichment of live feed organisms. The method used in the present study is based on a combination of a reverse-phase evaporation method for preparing liposomes and re-hydration of freeze-dried, empty liposomes. The liposomal membrane was made of soy phosphatydilcholine and was loaded with a highly concentrated free amino acids solution. Most of the liposomes produced were 2–8 μm in diameter and the FAA encapsulation efficiency was 42.6%. Two experiments simulating 2 hr of live food enrichment were used to evaluate the liposomes. The results showed the liposome did not disintegrate or aggregate when suspended in seawater and that only 9% of the FAA content of the liposomes was lost after 2 hr suspension. The developed method was easy and reliable, producing tens of grams of liposomes per batch.     

Calmodulin antagonists inhibit electron transport in photosystem II of spinach chloroplasts

Chlorpromazine, phenothiazine and trifluoperazine, known as calmodulin antagonists, inhibit electron transport in Photosystem II of spinach chloroplasts in concentrations from 20–500 μM. The inhibition site is located on the diphenyl carbazide to indophenol pathway in Tris-treated chloroplasts, indicating that water oxidation is not affected by these drugs. Ca²⁺ ions, bound to chloroplast membranes before the addition of calmodulin antagonists, can protect against inhibition up to 25% of the electron transport rate. In presence of A23187, the Ca²⁺-specific ionophore, Ca²⁺ ions provide less protection against inhibition by the 3 calmodulin antagonists used. A possible role of a calmodulin-like protein in spinach chloroplasts is postulated.