Preparation of biologically active mono-125I-insulin of high specific activity.

Pork insulin was labeled by the chloramine T technique (phosphate buffer 0.25 M; pH 7.5; EDTA 0.001 M; chloramine T: 0.2625 mg/ml; sodium metabisulfite 2.4 mg/ml) in a reaction volume of 50 microliters, adding chloramine T every 30 seconds twice (2.1 micrograms in 1 minute) and halting the reaction with 5 microliters metabisulfite. Three fractions were separated in preparative starch gel: F1 (mono-125I-insulin contaminated with cold insulin), F2 (mono-125I-insulin free from cold insulin), and F3 (di-125I-insulin). Insulins with low and high specific activity (iodine/insulin ratios 0.1/1 and 1/1 respectively) were prepared for study purposes, and quality was assessed by means of dose-response curves with antibodies and with liver cells. Specific activity of F2 as obtained from dose-response curves utilizing Scatchard's plot was 323 and 382 mCi/mg. Specific activity of F1 varied according to the extent of contamination with cold insulin. A reduction in the F2 B/F ratio was observed upon iodination with iodine/insulin ratios of 1/1 or in the neighborhood. The mass and immunoreactivity of F3, as well as its B/F ratios were constant, regardless of specific activity. The behavior with antibodies was ratified upon observations on uptake by liver cells and glucose consumption by isolated fat cells. In conclusion, F2 labeled with 0.1/1 iodine/insulin ratios was separated from cold insulin in preparative starch gel, thus increasing its specific activity (360 mCi/mg approximately) without alteration of its immunologic or biologic properties.

Isolation of liver cells with Ca2+ and K+ chelating agents. Biochemistry and cell morphology.

Cell morphology, glutamic pyruvic (GTP) and glutamic oxalacetic transaminases (GOT) concentrations, and the ability to produce glucose or urea from different substrates (pyruvate, alanine, fructose, lactate and glutamine) were studied in isolated mouse and rat liver cells in the presence of Ca2+ and K+ chelating agents (0.1 M sodium perchlorate and 0.027 M sodium citrate with 1 mg/ml bovine albumin; ionic strength: 0.198, pH: 7.4). The chelating agent is perfused through the portal vein of an in situ liver, at low pressure (8 ml/min) at 20 C for 15 min. Cell dispersion is obtained by cutting liver lobes and "massaging" the tissue with a plastic spatula. Wash and cell concentration may be obtained by sedimentation or centrifugation in Krebs III, glucose 150 mg %, improved with 0.16 M pyruvate, 0.1 M fumarate and 0.16 M glutamate. This procedure furnished 53.06 +/- 3.33 X 10(6) cells, which was highly significant (p less than 0.001) with respect to saline controls: 6.11 +/- 1.91 X 10(6). After staining with Papanicolaou, hematoxylin-eosin, and PAS, the cellular material obtained was classified optically into: normal isolated parenchymal liver cells, hepatocyte clumps, "burst" cells, normal blood or reticuloendothelial cells, cellular debris and non-cellular material. Cell morphology showed that a constant perfusion (8 ml/min) with a minimal mechanical treatment, 82.5% of the liver cells appears normal. Biochemical study showed that transaminases are indeed lost, but this loss is below the amount capable of effecting metabolic blockade (3/4 of transaminases remain in liver cells; GOT in cells: 692 +/- 218; GPT in cells. 264 +/- 94; GOT in supernatant: 152 +/- 29; GPT in supernatant: 79 +/- 12 mUI/10(6) cells, after recovering 60 min at 37 C) (means +/- SEM). Conversion of substrates (sodium pyruvate 10 mM, 20 mM D-L alanine, 10 mM fructose and 20 mM D-L sodium lactate) into glucose was statistically significant with respect to the baseline when the liver cells were isolated and recovered (rat liver cells, basal: 25.37 +/- 3.73; pyruvate: 54.04 +/- 7.98; DL-alanine: 62 +/- 10.07; fructose: 264.67 +/- 20.51; DL-lactate: 78.05 +/- 17.99 mmoles/10(6) cels, means +/- SEM). Urea production from 5 mM DL-glutamine was statistically highly significant to the basal with rat liver cell isolated and recovered (basal: 160.60 +/- 3.76; DL-glutamine: 608.47 +/- 16.15 mmoles/10(6) cells; means +/- SEM). The results obtained suggest that liver cells isolated with Ca2+ and K+ chelating agents used as described above are of value for biochemical studies.

Preparation of biologically active mono-125I-insulin of high specific activity.

Pork insulin was labeled by the chloramine T technique (phosphate buffer 0.25 M; pH 7.5; EDTA 0.001 M; chloramine T: 0.2625 mg/ml; sodium metabisulfite 2.4 mg/ml) in a reaction volume of 50 microliters, adding chloramine T every 30 seconds twice (2.1 micrograms in 1 minute) and halting the reaction with 5 microliters metabisulfite. Three fractions were separated in preparative starch gel: F1 (mono-125I-insulin contaminated with cold insulin), F2 (mono-125I-insulin free from cold insulin), and F3 (di-125I-insulin). Insulins with low and high specific activity (iodine/insulin ratios 0.1/1 and 1/1 respectively) were prepared for study purposes, and quality was assessed by means of dose-response curves with antibodies and with liver cells. Specific activity of F2 as obtained from dose-response curves utilizing Scatchards plot was 323 and 382 mCi/mg. Specific activity of F1 varied according to the extent of contamination with cold insulin. A reduction in the F2 B/F ratio was observed upon iodination with iodine/insulin ratios of 1/1 or in the neighborhood. The mass and immunoreactivity of F3, as well as its B/F ratios were constant, regardless of specific activity. The behavior with antibodies was ratified upon observations on uptake by liver cells and glucose consumption by isolated fat cells. In conclusion, F2 labeled with 0.1/1 iodine/insulin ratios was separated from cold insulin in preparative starch gel, thus increasing its specific activity (360 mCi/mg approximately) without alteration of its immunologic or biologic properties.

Isolation of liver cells with Ca2+ and K+ chelating agents. Biochemistry and cell morphology.

Cell morphology, glutamic pyruvic (GTP) and glutamic oxalacetic transaminases (GOT) concentrations, and the ability to produce glucose or urea from different substrates (pyruvate, alanine, fructose, lactate and glutamine) were studied in isolated mouse and rat liver cells in the presence of Ca2+ and K+ chelating agents (0.1 M sodium perchlorate and 0.027 M sodium citrate with 1 mg/ml bovine albumin; ionic strength: 0.198, pH: 7.4). The chelating agent is perfused through the portal vein of an in situ liver, at low pressure (8 ml/min) at 20 C for 15 min. Cell dispersion is obtained by cutting liver lobes and [quot ]massaging[quot ] the tissue with a plastic spatula. Wash and cell concentration may be obtained by sedimentation or centrifugation in Krebs III, glucose 150 mg

, improved with 0.16 M pyruvate, 0.1 M fumarate and 0.16 M glutamate. This procedure furnished 53.06 +/- 3.33 X 10(6) cells, which was highly significant (p less than 0.001) with respect to saline controls: 6.11 +/- 1.91 X 10(6). After staining with Papanicolaou, hematoxylin-eosin, and PAS, the cellular material obtained was classified optically into: normal isolated parenchymal liver cells, hepatocyte clumps, [quot ]burst[quot ] cells, normal blood or reticuloendothelial cells, cellular debris and non-cellular material. Cell morphology showed that a constant perfusion (8 ml/min) with a minimal mechanical treatment, 82.5

of the liver cells appears normal. Biochemical study showed that transaminases are indeed lost, but this loss is below the amount capable of effecting metabolic blockade (3/4 of transaminases remain in liver cells; GOT in cells: 692 +/- 218; GPT in cells. 264 +/- 94; GOT in supernatant: 152 +/- 29; GPT in supernatant: 79 +/- 12 mUI/10(6) cells, after recovering 60 min at 37 C) (means +/- SEM). Conversion of substrates (sodium pyruvate 10 mM, 20 mM D-L alanine, 10 mM fructose and 20 mM D-L sodium lactate) into glucose was statistically significant with respect to the baseline when the liver cells were isolated and recovered (rat liver cells, basal: 25.37 +/- 3.73; pyruvate: 54.04 +/- 7.98; DL-alanine: 62 +/- 10.07; fructose: 264.67 +/- 20.51; DL-lactate: 78.05 +/- 17.99 mmoles/10(6) cels, means +/- SEM). Urea production from 5 mM DL-glutamine was statistically highly significant to the basal with rat liver cell isolated and recovered (basal: 160.60 +/- 3.76; DL-glutamine: 608.47 +/- 16.15 mmoles/10(6) cells; means +/- SEM). The results obtained suggest that liver cells isolated with Ca2+ and K+ chelating agents used as described above are of value for biochemical studies.

[Noonan syndrome: differential diagnosis with Turner's syndrome]. / Síndrome de Noonan; diagnóstico diferencial con el síndrome de Turner.

Clinical and laboratory evidences assure an unequivocal identity to the syndrome described by Noonan. We believed that the terminology used by many authors has contributed to maintain confusion with Turner's syndrome from which it is clearly differenciated. The signology of both syndromes was confrontated in order to delineate the syndrome. Emphasis was made to point out the signs which are proper to each syndrome and the signs which are common to both of them stressing those that, occur with equal or significant difference. Two new signs are described in Noonan syndrome: alopecia of the hund portions of the eyebrows and keratosis rubra pilaris (Ulerythema ophriogenes).