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.     

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.     

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.     

Diet selection by European hares (Lepus europaeus) in the alpine zone of the Snowy Mountains, Australia

European hares (Lepus europaeus) are grazers and open grassland specialists that are replaced in mountain areas of their natural range in the northern hemisphere by browsing/intermediate feeding mountain hares (Lepus timidus), but in their introduced range in the southern hemisphere, occupy the alpine zone. We used micro-histological identification of plant fragments and germination of seeds in faecal pellets of L. europaeus from the Snowy Mountains, Australia, to determine diet. We asked whether diet shifted and/or diet breadth expanded in response to seasonally reduced food availability, particularly during winter. If so, did the constraints of food availability in the alpine zone lead to the diet mirroring that of L. timidus in its native alpine habitat. The diet of L. europaeus was dominated by grasses, herbs and shrubs. The main diet items in summer were grasses (70 %) and herbs (28 %). Grasses declined in the diet between summer and autumn when herbs increased to co-dominance, with a further change after establishment of the winter snowpack to a greater preponderance of shrubs (43 % compared with a maximum of 3 % in snow-free months). L. europaeus selected a wider range of plants in winter (59 species compared with 39 in summer) and diet was significantly more variable in winter than in autumn or summer (and in autumn than summer). We concluded that the persistence of L. europaeus in alpine areas of the southern hemisphere is testament to their ability to expand their dietary breadth to occupy mountain climatic zones normally occupied by L. timidus.     

IQ-motif peptides as novel anti-microbial agents

The IQ-motif is an amphipathic, often positively charged, α-helical, calmodulin binding sequence found in a number of eukaryote signalling, transport and cytoskeletal proteins. They share common biophysical characteristics with established, cationic α-helical antimicrobial peptides, such as the human cathelicidin LL-37. Therefore, we tested eight peptides encoding the sequences of IQ-motifs derived from the human cytoskeletal scaffolding proteins IQGAP2 and IQGAP3. Some of these peptides were able to inhibit the growth of Escherichia coli and Staphylococcus aureus with minimal inhibitory concentrations (MIC) comparable to LL-37. In addition some IQ-motifs had activity against the fungus Candida albicans. This antimicrobial activity is combined with low haemolytic activity (comparable to, or lower than, that of LL-37). Those IQ-motifs with anti-microbial activity tended to be able to bind to lipopolysaccharide. Some of these were also able to permeabilise the cell membranes of both Gram positive and Gram negative bacteria. These results demonstrate that IQ-motifs are viable lead sequences for the identification and optimisation of novel anti-microbial peptides. Thus, further investigation of the anti-microbial properties of this diverse group of sequences is merited.