The reaction of chemical probes with the erythrocyte membrane

Summary Trinitrobenzenesulfonate (TNBS), fluorodinitrobenzene (FDNB) and suberimidate have been reacted with intact human erythrocytes. TNBS does not penetrate the cell membrane significantly at 23 °C in bicarbonate-NaCl buffer, pH 8.6, as estimated by the labeling of the N-terminal valine of hemoglobin. Hence, under these conditions it can be used as a vectorial probe. However, at 37 °C, especially in phosphate buffer, at pH 8.6, TNBS does penetrate the cell membrane. FDNB and suberimidate both penetrate the erythrocyte membrane. The time course reaction of TNBS with intact erythrocytes over a 24-hr period at 23 °C is complex and shows transition zones for both membrane phosphatidylethanolamine (PE) and membrane proteins. No significant cell lysis occurs up to 10 hr. The fraction of total PE or phosphatidylserine (PS) which reacts with TNBS by this time period can be considered to be located on the outer surface of the cell membrane. Under these conditions it can be shown that 10 to 20% of the total PE and no PS is located on the outer surface of the membrane and hence these amino phospholipids are asymmetrically arranged. The pH gradient between the inside and outside of the cell in our system is 0.4 pH units. Nigericin has no effect on the extent of labeling of PE or PS by TNBS. Isotonic sucrose gives a slight enhancement of the labeling of PE by TNBS. Hence, the inability of PE and PS to react with the TNBS is considered not due to the inside of the cell having a lower pH. The extent of reaction of TNBS with PE is not influenced by changing the osmolarity of the medium or by treatment of cells with pronase, trypsin, phospholipase A or phospholipase D. However, bovine serum albumin (BSA) does protect some of the PE molecules from reacting with TNBS.Cells treated with suberimidate were suspended in either isotonic NaCl or in distilled water. In both cases the suberimidate-treated cells became refractory to hypotonic lysis. Pretreatment of cells with TNBS did not prevent them from interacting with suberimidate and becoming refractory to lysis. However, pretreatment of cells with the penetrating probe FDNB abolished the suberimidate, effect. Electron-microscopic analysis of the cells showed a continuous membrane in the case of cells suspended in isotonic saline. The cells suspended in water did not lyse but their membranes had many large holes, sufficient to let the hemoglobin leak out. Since the hemoglobin did not leak out we know that the hemoglobin is cross-linked into a large supramolecular aggregate.     

Otolith patterns of rockfishes from the northeastern pacific

Sagitta otolith shape was analysed in twenty sympatric rockfishes off the southern California coast (Northeastern Pacific). The variation in shape was quantified using canonical variate analysis based on fifth wavelet function decomposition of otolith contour. We selected wavelets because this representation allow the identifications of zones or single morphological points along the contour. The entire otoliths along with four subsections (anterior, ventral, posterodorsal, and anterodorsal) with morphological meaning were examined. Multivariate analyses (MANOVA) showed significant differences in the contours of whole otolith morphology and corresponding subsection among rockfishes. Four patterns were found: fusiform, oblong, and two types of elliptic. A redundancy analysis indicated that anterior and anterodorsal subsections contribute most to define the entire otolith shape. Complementarily, the eco‐morphological study indicated that the depth distribution and strategies for capture prey were correlated to otolith shape, especially with the anterodorsal zone. J. Morphol. 276:458–469, 2015. © 2014 Wiley Periodicals, Inc.     

Seasonal Differences in Hypoxia Tolerance in Gulf Killifish, Fundulus Grandis (Fundulidae)

Many estuarine habitats are characterized by episodes of hypoxia, the frequency and severity of which may vary seasonally. Accordingly, resident fish species may show seasonal differences in their capacity to tolerate hypoxia. We have tested this hypothesis in the gulf killifish, Fundulus grandis, sampled from the Lake Pontchartrain estuary (Louisiana) at different times of the year. We measured 2 indicators of hypoxia tolerance, the frequency of aquatic surface respiration (ASR) during gradual reduction in dissolved oxygen (D.O.) and survival time during severe hypoxic stress, and found both to be significantly affected by season. Fish collected during the summer did not engage in ASR until the D.O. concentration dropped to values lower than that required to elicit ASR by fish collected during other seasons. Laboratory acclimation of fish to low oxygen did not change the relationship between ASR behavior and D.O., suggesting that the observed seasonal effect on ASR was not simply due to previous exposure of summer fish to environmental hypoxia. Furthermore, fish collected during the summer and winter had significantly longer survival times during exposure to severe hypoxia than fish collected during the fall. Survival analysis indicated that the condition of fish was positively associated with survival time, and seasonal variation in condition accounted for about half of the observed difference between survival times of fish collected during the summer and fall. Seasonal variation in ASR and survival, when taken together, demonstrate that hypoxia tolerance in F. grandis may be subject to acclimatization. An increase in hypoxia tolerance during the summer could increase survivorship of fish when exposed to elevated temperatures and low oxygen concentrations which prevail during the summer months.