 toy line.  And before I do, I write a letter to my 8th grade self.
In the fifties they were for squares. In the eighties, punks. And most recently they've been the domain of the skinny-jeaned. But these days you don't need a subculture membership card to get away with thick frames. Though a proclivity to drop Gore Vidal's name into casual conversation helps, all that's really required is commitment. If you have what it takes to own the look, these bookish beauties can give your face a big lift.
1. To pull off Clark Kents, it helps to have Superman bone structure. (Tiny eyes and high cheekbones tend to get lost behind heavier glasses.) The stronger the features the better, but even if you don't have a chiseled jaw, these frames bring plus-size noses and expansive foreheads into proportion.
2. They used to come exclusively in black, precluding anyone without dark hair from wearing them. Today's blonde and brown frames allow guys with fairer complexions to bulk up too.
3. Thick specs can tame a protruding brow line. Just make sure the top of the frame sits squarely on your brows—not above them.What caused the formation of city-states?

For context I am perhaps most interested in the Italian city-states of the medieval and renaissance periods. Though insight into others like the Hanseatic League could be interesting.

What caused these entites to form in the first place? Did certain conditions coalesce to support their formation? Did geography protect them from being gobbled up by larger neighbours? 

I find city-states interesting in general so information about other examples would be welcome too.Former NIGHTWISH vocalist TARJA TURUNEN has been in Austin, TX working on her upcoming album, slated for release sometime in August or September. The name of the album will be revealed later this month. Keeping fans informed, she posted an update revealing the title of three new songs: ‘Never Enough’, ‘Into The Sun’, and ‘Medusa’.
Other song titles set to appear on the album are ‘Lucid Dreamer’, ‘Neverlight’, and ‘500 Letters’. Tarja recently filmed a lyric video for ‘Never Enough’ and is expected to premiere soon.
Separation of the Half -Molecular Weight Nonidentical Subunits of Pigeon Liver Fatty Acid Synthetase by Affinity Chromatography*

SUMMARY Two half-molecular weight subunits of 4’-phospho[14C]pan-tetheine-labeled pigeon liver fatty acid synthetase have been separated and shown to be nonidentical. An affinity chro-matographic column containing e-aminocaproylpantetheine bound to cyanogen bromide-activated Sepharose via the E-amino group was used to achieve this separation. The subunit containing 4’-phospho(14C]pantetheine and /3-keto-acyl thioester reductase activity is only slightly retarded on this column, whereas the subunit containing acetyl coenzyme A transacylase activity is strongly adsorbed. The latter subunit is eluted from the column with 0.1 M phosphate buffer at pH 10. Each subunit is then purified free of small amounts of the other subunit. This is accomplished by subjecting each to conditions which effect fatty acid synthetase forma-tion. Subsequent sucrose density gradient centrifugation then separates the pure half-molecular weight subunits containing transacylase or P-ketoacyl thioester reductase activity from the small amount of fatty acid synthetase complex. purified under appro-priate


SUMMARY
Two half-molecular weight subunits of 4'-phospho[14C]pantetheine-labeled pigeon liver fatty acid synthetase have been separated and shown to be nonidentical.
An affinity chromatographic column containing e-aminocaproylpantetheine bound to cyanogen bromide-activated Sepharose via the E-amino group was used to achieve this separation.
The subunit containing 4'-phospho(14C]pantetheine and /3-ketoacyl thioester reductase activity is only slightly retarded on this column, whereas the subunit containing acetyl coenzyme A transacylase activity is strongly adsorbed.
The latter subunit is eluted from the column with 0.1 M phosphate buffer at pH 10. Each subunit is then purified free of small amounts of the other subunit. This is accomplished by subjecting each to conditions which effect fatty acid synthetase formation.
Subsequent sucrose density gradient centrifugation then separates the pure half-molecular weight subunits containing transacylase or P-ketoacyl thioester reductase activity from the small amount of fatty acid synthetase complex. Recombination of the purified subunits under appropriate conditions yields enzymatically active fatty acid synthetase complex.
The pigeon liver fatty acid synthetase complex dissociates reversibly' into two subunits of approximately equal molecular weight (1,2). However, the separation of these subunits from one another had not been accomplished prior to this communication. Indeed, the similarity of the masses and the mass to charge ratios of the two half-molecular weight subunits is such that the question of their identity or nonidentity was unanswered until it was shown that only one phosphopantetheine group is present, per intact fatty acid synthet.ase molecule ( likely the subunits would have unequal affinity for an external analog of the internal substrate family of acyl 4'.phosphopantetheines.
If so, the separation of nonidentical subunits could then be proven by assay of each for the partial reactions of fatty acid synthesis.
Two partial reactions of fatty acid synthesis (Equations 1 and 2) were used in the present investigation to follow the separation of the half-molecular weight subunits of pigeon liver fatty acid synthetase. II 0 We chose as an affinity ligand in this investigation the substrate analog e-aminocaproylpantetheine bound to Sepharose by means of the amino group.
This ligand is analogous to the intermediate caproyl-4'.phosphopantetheine (4). Sepharose e-amino-n-caproic acid was prepared according to the method of Larsson and Xosbach (5) from cyanogen bromide-activated Sepharose (6). Crystalline pantetheine was obtained from Sigma Chemical Co., and it was reduced with sodium amaIgam to pantetheine prior to use. The pH of the reaction was kept, below 7 by the addition of Dowex 50-H+.
Sepharose e-aminocaproylpantetheine was prepared by the method of Cuatrccasas (7) but with the -SH compound in the solvent and the carbonyl compound in the stationary phase. An amount of ethyldi-methylamino~n-propylcarbodiimide sufficient to enable the binding of only 1.5 pmoles of pantetheine to 1 g of gel (wet weight) was used. This is less than the amount given by Cuatrecasas.
Larger amounts of the carbodiimide lead to excessive nonspecific (ionic) binding of fatty acid synthetase subunits. lJou1~1 pant.etheine was assayed by the Ellman method after hydrolyzing the washed Sepharose-e-aminocaproylpantetheine in 0.1 N KOH for 15 min at, room temperature.
The Sepharose e-aminocaproylpantethcinc was washed with 1 liter of dist.illed water and then with 150 ml of buffer (pH 8.7) cont,aining 0.1 M potassium phosphate, 0.1 hf Tris, and 0.0025 hf fi-mercaptoethanol or dithiothreitol. The final pII of the solution was 8.7. The Sepharose substrate analog was then packed in an ice-salt water-jacketed column (3 mm x 15 cm). ['4C]I'antetheit~e-labcled fatty acid synthetase (10 mg/0.5 ml) prepared as described previously (8) was dissociated by dialyzing 4 to 5 hours at 0" against 5 mM Tris, 35 rnhr glycine, 1 m&f EDTA, and 5 mM P-mercaptoethanol or dithiothreitol(1). The dialyzed, dissociated fatty acid synthetase was diluted with the same buffer to 2 ml and then loaded onto the Sepharose e-aminocaproylpantetheine column at O-1". Elution was carried out with at least 50 ml of a 0.1 M potassium phosphate-O.1 M Tris buffer (pH 8.5) containing 5 mM fl-mercaptoethanol at -1.0-O" and then at 25" with the same buffer adjusted to pH 10 with ammonia.
Loading and elution were carried out at a rate of 3 ml per hour and l-ml fractions were collected.
The fractions were monitored for ultraviolet light absorption at 280 nm. &Ketoacyl thioester reductase in each fraction was assayed spectrophotometrically, with N-acetyl-S-acetoacetyl cysteamine (Sigma) and TPNH as substrates by the method of Kumar et al. (9). Acetyl-CoA-pantetheine transacylase activity was measured radiochemically in a system consisting of 0.03 to 0.05 pg of protein, 3.2 nmoles of [Wlacetyl-CoA (4000 cpm per nmole), and 0.34 pmole of pantetheine in 0.1 ml of 0.2 M