Characterization of phiA, a gene essential for phialide development in Aspergillus nidulans

We have previously identified genes and proteins involved in the fungal response to the Streptomyces-produced antibiotics, bafilomycin B1 and concanamycin A, known inhibitors of V-ATPases. Using mRNA differential display we identified an Aspergillus nidulans gene with 30-fold up-regulated expression in the presence of bafilomycin. This gene, here denoted phiA, and its gene product, were further characterized by targeted gene disruption and immunohistochemistry. Phenotypically, the phiA mutation resulted in reduced growth and severely reduced sporulation. The abnormality could be traced to the phialides, which divided several times instead of forming a single flask-shaped cell. The importance of phiA for phialide and conidium development was supported by immunohistochemistry experiments that showed the protein to be mainly present in these two cell types. Attempts to relate phiA to inhibition of V-ATPases did not result in unambiguous conclusions, but suggest the possibility that changed expression of phiA is correlated with growth arrest caused by inhibited V-ATPases.     

Hyperhydration induced by glycerol ingestion: hormonal and renal responses

The simultaneous time courses of hydromineral hormones (renin-aldosterone system, arginine vasopressin, and atrial natriuretic peptide) and renal responses were examined during and after the completion of hyperhydration induced by glycerol and fluid ingestion. Eight healthy young male Caucasian subjects participated in two separate trials, each including three consecutive phases in a thermoneutral environment. Phases 1 and 3 involved a 90-min period at rest, while phase 2 involved a 120-min period at rest designed to provide either (i) euhydration (control trial) or (ii) hyperhydration induced by ingestion of glycerol (1.1 g/kg body mass) and fluid (21.4 mL/kg body mass). During the 2-h time period of glycerol and fluid ingestion, urine flow, urine osmolality, and plasma levels of hydromineral hormones remained at basal values. In contrast, after hyperhydration completion during phase 3, the diuresis increased markedly together with a dilution of the urine (p < 0.05) while hormonal responses did not change. These results indicate significant differences in renal responses during and after hyperhydration completion and suggest that these changes are independent of fluid-regulating hormonal responses.     

Investigation of a failed DNA assay leads to identification of an unexpected contaminant in a commonly used chromatography buffer

For mammalian cell-derived recombinant biotherapeutics, controlling host cell DNA levels below a threshold is a regulatory requirement to ensure patient safety. DNA removal during drug substance manufacture is accomplished by a series of chromatography-based purification steps and a qPCR-based analytical method is most used to measure DNA content in the purified drug substance to enable material disposition. While the qPCR approach is mature and its application to DNA measurement is widespread in the industry, it is susceptible to trace levels of process-related contaminants that are carried forward. In this study, we observed failures in spike recovery studies that are an integral component of the qPCR-based DNA testing, suggesting the presence of an inhibitory compound in the sample matrix. We generated hypotheses around the origin of the inhibitory compound and generated multiple sample matrices and deployed a suite of analytical techniques including Raman and NMR spectroscopy to determine the origin and identity of the inhibitory compound. The caustic wash step and depth filter extractables were ruled out as root causes after extensive experimentation and DNA testing. Subsequently, 2-(N-morpholino)ethanesulfonic acid (MES), a buffer used in the chromatography unit operations, was identified as the source of the contaminant. A 500-fold concentration followed by Raman and NMR spectroscopy analysis revealed the identity of the inhibitory compound as polyvinyl sulfone (PVS), an impurity that originates in the MES manufacturing process. We have implemented PVS concentration controls for incoming MES raw material, and our work highlights the need for rigor in raw material qualification and control.     

Activities of pulmonary phospholipases of fetal rats. Variations during development]

Turnover of adult rat lung phospholipids implies intervention of phospholipases. This work clearly demonstrates: There is in fetal or adult rat lung an inactive form of phospholipase that is convertible to an active form by the action of lysed platelets. An increase of both active and inactive forms of the fetal enzyme with gestational age. The fact that an important part of these activities, at the time of birth, are in the inactive form implies a control mechanism affecting levels of each form of lung phospholipases. These data are discussed in relation to the possible role of the lung phospholipases in Respiratory Distress Syndrome.     

Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.) : Orientation of Active Site and Activation by Ca and Mg

Polyphosphoinositide-specific phospholipase C activity was present in plasma membranes isolated from different tissues of several higher plants. Phospholipase C activities against added phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP₂) were further characterized in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticum aestivum L. cv Drabant) seedlings. In right-side-out (70-80% apoplastic side out) plasma membrane vesicles, the activities were increased 3 to 5 times upon addition of 0.01 to 0.025% (w/v) sodium deoxycholate, whereas in fractions enriched in inside-out (70-80% cytoplasmic side out) vesicles, the activities were only slightly increased by detergent. Furthermore, the activities of inside-out vesicles in the absence of detergent were very close to those of right-side-out vesicles in the presence of optimal detergent concentration. This verifies the general assumption that polyphosphoinositide phospholipase C activity is located at the cytoplasmic surface of the plasma membrane. PIP and PIP₂ phospholipase C was dependent on Ca²⁺ with maximum activity at 10 to 100 μm free Ca²⁺ and half-maximal activation at 0.1 to 1 μm free Ca²⁺. In the presence of 10 μm Ca²⁺, 1 to 2 mm MgCl₂ or MgSO₄ further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mm Ba²⁺, Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, and Zn²⁺) inhibited the enzyme activity. The stimulatory effect by Mg²⁺ was observed also when 35 mm NaCl was included. Thus, the PIP and PIP₂ phospholipase C exhibited maximum in vitro activity at physiologically relevant ion concentrations. The plant plasma membrane also possessed a phospholipase C activity against phosphatidylinositol that was 40 times lower than that observed with PIP or PIP₂ as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca²⁺, with maximum activity at 1 mm CaCl₂, and could not be further stimulated by Mg²⁺.     

In vivo effect of diosmin on carrageenan and CCl4-induced lipid peroxidation in rat liver microsomes

The aim of this study was to compare the protective effect of a flavonoid, the 3′5,7-trihydroxy-4′-methoxyflavone 7-rutinoside or diosmin, on liver microsomal lipid peroxidation induced in rats by either carbon tetrachloride or carrageenan. Thirty rats were divided into five groups. Group 1 received no chemical product and was considered as control. Groups 2 and 3 received either an intraperitoneal injection of carrageenan or carbon tetrachloride 48 or 24 hours before killing, respectively. Groups 4 and 5 were treated first with an intraperitoneal injection of diosmin and then by carrageenan (group 4) or carbon tetrachloride (group 5) 48 or 24 hours before killing, respectively. The lipoperoxidant effect of carrageenan and carbon tetrachloride was demonstrated by both significant decreases in polyunsaturated fatty acids, principally 20:4 (n− 6) (p < 0.05) and of vitamin A (p < 0.05) in groups 2 and 3. With diosmin treatment, only thiobarbituric acid reactive substances significantly decreased in group 4, whereas vitamin A level increased. These results could suggest that the effect of diosmin differs with the choice of chemical product used; it seems a better antioxidant against products inducing inflammation. © 1996 John Wiley & Sons, Inc.     

Infant cannibalism in wild white‐faced capuchin monkeys

Cannibalism has been observed in a variety of animal taxa; however, it is relatively uncommon in primates. Thus, we rely heavily on case reports of this behavior to advance our understanding of the contexts under which it occurs. Here, we report the first observation of cannibalism in a group of wild white‐faced capuchin monkeys (Cebus imitator). The subject was a dead infant, estimated to be 10 days old, and the probable victim of infanticide. Consumption of the corpse was initiated by a 2‐year‐old male (second cousin of the infant), though it was eventually taken over and monopolized by the group''s alpha female (grandaunt of the infant). Although most group members expressed interest in the corpse (sniffing, touching, and threatening it), no others made an attempt to consume it. Given that this is the only observation of cannibalism recorded in over 37 years of study on this population, we consider it to be a rare behavior in this species. This detailed record contributes new data, which, when combined with other reports within and across species and contexts, enables the evaluation of adaptive explanations of cannibalism.