A Note on the Purification of Alcian Blue

Alcian blue 8GX is a copper phthalocyanin dye that shows a high degree of specificity for polyanionic substances such as hyaluronic acid, sialic acid and the chondroitin sulfates. This dye has proved useful for both histochemical and electrophoretic staining of these substances. The Biological Stain Commission has recently begun to certify Alcian blue (Schenk 1981). Commercially available lots contain approximately 50% dye. The remaining constituents have been identified as primarily boric acid, as well as sulfates and dextrins (Scott 1972, Horobin and Goldstein 1972). Horobin and Goldstein (1972) have pointed out that these contaminants may adversely affect staining in the critical electrolyte concentration procedure. Scott (1972), while not ascribing any adverse effects to the presence of boric acid, recommends its removal by differential precipitation with acetone. In this procedure one part of a 2-5% aqueous solution of the dye is added to 5-10 parts of acetone. The precipitated dye is approximately 80% pure. While this method is relatively simple, it does have several drawbacks. Low concentrations of Alcian blue (i.e., 2%) must be used to obtain purities near 80%. Thus a minimum of 250 ml of acetone is needed to purify 1 gram of dye. Furthermore, Horobin and Goldstein (1972) have reported that contamination by dextrin or unknown organic substances (detergent?) interferes with precipitation of the dye enough to make purification by Scott's method impossible. When difficulty in the precipitation of Alcian blue by Scott's method was encountered, the following simple method for the purification of the dye was developed.     

Isopycnography of intact cells — V: The kinetics of the initiation of DNA synthesis in E. coli undergoing nutrient shift-up

Manipulation of the ribosome content of E. coli by means of a nutrient shift-up leads to predictable changes in cellular specific gravity. Thus whole-cell pycnography can be used to monitor the proliferative status of the rRNA loci which cluster closely about the genetic origin of DNA synthesis. In this manner the rate of initiating new rounds of genome replication was followed during an upshift. The results indicate that after a short lag initiation of new rounds abruptly and completely shifts to the rate appropriate to the enriched conditions.     

A comparative binding of platinum anti-tumour compounds to plasma proteins in the rat (in vivo) and mouse (in vitro).

Plasma protein binding of 195mPt-labelled cisplatin, carboplatin and iproplatin has been studied in vivo in rat and in vitro in mouse, using both electrophoresis and trichloroacetic acid precipitation. After intravenous injection plasma clearance rates were biphasic for all 3 compounds, (t1/2 alpha, 13-17 min) but cisplatin was retained thereafter longer than the others. By 5 min, gel electrophoresis showed protein labelling with all 3 drugs but none involved low mol.wt. proteins (< 16 kDa). At 2 h a notable proportion of the protein bound platinum was associated with the latter components. There was a general resemblance between the distribution patterns of cisplatin and carboplatin whereas iproplatin showed a persistent retention of the label with time to higher mol. wt. proteins. From in vitro incubation with mouse plasma, rates of interaction respectively were cisplatin t1/2 alpha, 35 min, beta 8 h, carboplatin t1/2, 44 h and iproplatin t1/2, 104 h. By electrophoresis the protein bound fraction pattern (1 h) was again similar for cisplatin and carboplatin with virtually no binding to low mol. wt. proteins. After 24 h these were now involved to a high degree (40%). Iproplatin showed relatively marked binding to proteins of higher mol. wt. but no transfer with time to the low mol. wt. protein zone. A possible explanation is the need for in vivo metabolism for this compound as manifest in the rat. It is suggested that the significance of interaction with low mol. wt. proteins merits further investigation in relation to the antitumour and toxicological actions of these drugs.     

The ascidian sperm reaction: Ca2+ uptake in relation to H+ efflux

During the ascidian sperm reaction the single large cylindrical mitochondrion which lies next to the nucleus in the head swells, becomes spherical, and migrates along the tail to be lost when it reaches the end. This sequence is initiated by eggs, egg water, high pH, low Na⁺, or the ionophore X537A. Accompanying the sperm reaction induced by low Na⁺ are H⁺ efflux and Ca²⁺ influx in a ratio of near 100:1 as determined by ⁴⁵Ca²⁺ and atomic absorption analysis. Simultaneous pH and Ca²⁺ electrode measurements suggest that the movement of H⁺ begins 10–13 sec before the movement of Ca²⁺. Ca²⁺ uptake can be inhibited by verapamil without affecting H⁺ efflux or the sperm reaction. Acid release and Ca²⁺ uptake are proportional to the initial pH of the medium when the reaction is triggered by high pH. Acid release initiated by low Na⁺ is proportional to Ca²⁺ concentrations above 2 mM. H⁺ and Ca²⁺ movements differ in magnitude, kinetics, and inhibition by verapamil, thus suggesting that H⁺ is probably not exchanged for Ca²⁺. Instead we propose that loss of H⁺ triggers the uptake of Ca²⁺, which initiates the sperm reaction.     

A proposed mechanism relating the antitumor behavior of cis-platinum amine complexes to their inhibition of a model enzyme

The twenty-four hour inhibition of m-malate dehydrogenase (E.C. 1.1.1.37) by various complexes of cis-platinum(II) and cis-platinum(IV) was measured as a function of the platinum concentration. It was observed that increased alkylation of the amine groups of Pt(II) and to a lesser degree of Pt(IV) decreased the activity consistently. It was also observed that the Pt(IV) analogues inhibit the enzyme to about an order of magnitude greater than the Pt(II) complexes. These phenomena will be interpreted.     

Menstrual cycle phase and oral contraceptive effects on triglyceride mobilization during exercise.

We examined the effects of menstrual cycle phase and oral contraceptive (OC) use on triglyceride mobilization during 90 min of rest and 60 min of leg ergometry exercise at 45 and 65% peak O(2) uptake (Vo(2 peak)) in eight moderately physically active, eumenorrheic women (24.8 +/- 1.2 yr). Subjects were tested during the follicular phase (FP) and the luteal phase (LP) before OC use and during the inactive phase (IP) and high-dose phase (HP) after 4 complete mo of OC use. Glycerol rate of appearance (R(a)), a measure of triglyceride mobilization, was determined in a 3-h postabsorptive state using a primed constant infusion of [1,1,2,3,3-(2)H]glycerol. Before OC use (BOC), there were no significant differences between FP and LP in any of the variables studied. Dietary composition, exercise patterns, plasma glycerol concentrations, growth hormone concentrations, and exercise respiratory exchange ratio did not change with OC use. However, 4 mo of OC use significantly (P < 0.05) increased glycerol R(a) in HP during exercise at 45% Vo(2 peak) (6.2 +/- 0.2, 6.5 +/- 0.4, and 7.7 +/- 1.1 micromol.kg(-1).min(-1) for BOC, IP, and HP, respectively) and in IP and HP at 65% Vo(2 peak) (6.6 +/- 0.1, 8.2 +/- 0.6, and 8.1 +/- 0.7 micromol.kg(-1).min(-1) for BOC, IP, and HP, respectively). Plasma cortisol concentrations were significantly higher with OC use at rest and during exercise at 45 and 65% Vo(2 peak). In summary, although fluctuations of endogenous ovarian steroids have little effect on triglyceride mobilization, the synthetic ovarian steroids found in OCs increase triglyceride mobilization and plasma cortisol concentrations in exercising women. We conclude that the hierarchy of effects of ovarian steroids and their analogs on triglyceride mobilization in exercising women is as follows: energy flux > OC use > recent carbohydrate nutrition, menstrual cycle effects.     

Punishment is strongly motivated by revenge and weakly motivated by inequity aversion

There are two broad functional explanations for second-party punishment: fitness-leveling and deterrence. The former suggests that people punish to reduce fitness differences, while the latter suggests that people punish in order to reciprocate losses and deter others from inflicting losses on them in the future. We explore the relative roles of these motivations using a pre-registered, two-player experiment with 2426 US participants from Amazon Mechanical Turk. Participants played as the “responder” and were assigned to either a Take or Augment condition. In the Take condition, the “partner” could steal money from the responder's bonus or do nothing. In the Augment condition, the partner could augment the responder's bonus by giving them money at no cost to themselves or do nothing. We also manipulated the responders' starting endowments, such that after the partner's decision, responders experienced different payoff outcomes: advantageous inequity, equality, or varying degrees of disadvantageous inequity. Responders then decided whether to pay a cost to punish the partner. Punishment was clearly influenced by theft and was most frequent when theft resulted in disadvantageous inequity. However, people also punished in the absence of theft, particularly when confronted with disadvantageous inequity. While the effect of inequity on punishment was small, our results suggest that punishment is motivated by more than just the desire to reciprocate losses. These findings highlight the multiple motivations undergirding punishment and bear directly on functional explanations for the existence of punishment in human societies.