Polypeptide composition and organization of the sea urchin extraembryonic hyaline layer

The protein composition and organization of the sea urchin extraembryonic hyaline layer was examined. Hyalin and a polypeptide of 45 kilodaltons (kDa) were present in hyaline layers isolated from 1-h-old embryos through to the pluteus larva stage. In contrast, several polypeptide species ranging in size from 175 to 32 kDa either decreased in amount or disappeared from the layer as embryonic development proceeded. Concomitant with the changes in composition, hyaline layers became progressively more refractory to dissolution by washing in Ca2+, Mg2+-free seawater. Incubation of intact layers, isolated from 1-h-old embryos, with proteinase K resulted in the selective digestion of hyalin and was accompanied by release of the 45-kDa polypeptide from the layers. Washing intact layers in 20 mM Tris (pH 8.0) also resulted in the selective removal of hyalin and the 45-kDa polypeptide. The Ca2+-precipitable protein hyalin, alone among the hyaline layer polypeptides, bound the Ca2+-antagonist ruthenium red. These results suggest a structural organization within the hyaline layer that is both heterogenous and dynamic throughout embryonic development.Key words: hyaline layer, composition, organization, development.

In vivo somatostatin, vasopressin, and oxytocin synthesis in diabetic rat hypothalamus

The in vivo labeling of somatostatin-14, somatostatin-28, arginine vasopressin, and oxytocin was studied in rat hypothalamus after third ventricular administration of [35S]cysteine to streptozotocin-diabetic and normal rats. Immunoreactive somatostatin levels in hypothalamus were unaffected by diabetes, as was the incorporation of [35S]cysteine into hypothalamic somatostatin-14 and somatostatin-28. In contrast, immunoreactive vasopressin levels in hypothalamus and posterior pituitary (and oxytocin levels in posterior pituitary) were below normal in diabetic rats. Moreover, [35S]cysteine incorporation into hypothalamic vasopressin and oxytocin (probably mainly in the paraventricular nucleus because of its proximity to the third ventricular site of label injection) was significantly above normal. The increments in vasopressin and oxytocin labeling were reversed by insulin administration. In vivo cysteine specific activity and the labeling of acid-precipitable protein did not differ between normal and diabetic animals; effects of diabetes on vasopressin and oxytocin labeling were therefore not caused by simple differences in cysteine specific activity. These results suggest that diabetes 1) does not influence the production of somatostatin peptides in hypothalamus but 2) stimulates the synthesis of vasopressin and oxytocin. For vasopressin at least, the increase in synthesis may be a compensatory response to the known increase in its secretion that occurs in uncontrolled diabetes.

Regulation of lipoprotein lipase synthesis in 3T3-L1 adipocytes by cachectin Further proof for identity with tumour necrosis factor

We investigated the mechanism by which the endotoxin-induced macrophage secretory protein cachectin is able to suppress the activity of lipoprotein lipase in 3T3-L1 adipocytes. The loss in activity results from an effect on the synthesis of the enzyme, as determined by a decreased incorporation of [35S]methionine into immunoprecipitable lipoprotein lipase. The results were nearly identical whether crude conditioned medium or a highly purified preparation was utilized as a source of cachectin. [35S]Methionine incorporation into acid-precipitable protein was minimally affected by purified cachectin, suggesting that the suppression of the lipoprotein lipase was not due to a general suppression of protein synthesis. These results, taken together with our previous work, provide additional evidence that cachectin and tumour necrosis factor are functionally identical.

Deproteinization Effects on Homogenized Leaf Cured (HLC) Products

The HLC deproteinized product [depro] with 34 % of its original dry weight removed as heat precipitable protein fractions, shows a reduction in the levels of most of the cured product constituents and the major pyrolyzate constituents while the levels of polycyclic aromatic hydrocarbons and volatile nitrosamines in the pyrolyzates from the HLC deproteinized product were increased. This indicates that the precursors of the polycyclic aromatic hydrocarbons in the pyrolyzate from the HLC deproteinized product were not removed with the protein precipitates. Another possibility is that some constituents in the protein precipitates that were removed from the HLC deproteinized product may have had an inhibitory effect toward the formation of the polycyclic aromatic hydrocarbons in the pyrolyzates of the HLC control and flue-cured reference during their pyrolysis, since the protein precipitates were not removed from these products. Generally, the levels of polycyclic aromatic hydrocarbons from the pyrolyzates of the HLC products were higher than those from the pyrolyzates of the flue-cured reference. This could be a result of the tobacco maturity or curing methods. The level of the volatile nitrosamines in the pyrolyzates of the HLC control was lower than those levels in the pyrolyzates of the HLC deproteinized and flue-cured reference products. This could be due to pyrolytic interaction between some constituents in the sample and the protein precipitate which was not removed from the HLC control product. Additional work is needed to clarify these differences. A reduction in the level of solanesol was evident in the HLC deproteinized product probably due to the association of solanesol with the chloroplastic protein precipitate that was removed from that product. In addition, the level of solanesol was highest in the flue-cured reference, which is in agreement with previous reports that solanesol concentration increases with tobacco maturity. This report demonstrates that HLC can be used to manipulate the chemical composition of tobacco. The levels of some major constituents were decreased while the levels of polycyclic aromatic hydrocarbons were increased in the pyrolyzate from the same tobacco product.

Abnormal Fibrin Monomer Polymerization In Patients With Lupus Erythematosus Systemicus (LES)

Fibrin monomer polymerization was studied in 10 out of 44 patients with LES, selected for their prolonged thrombin time (patients 24.6 ± 1.9 sec; normal controls 15.0 ± 0.5 sec). One of these patients had also a lupus-like blood clotting inhibitor. The level of fibrinogen measured as thrombin clottable protein was 407.5 ± 62.7 mg/dl, and as heat precipitable protein 325.8 ± 49.3 mg/dl. Fibrin monomer polymerization with thrombin assayed as the increment in O.D., showed in 5 patients a delay of two minutes compared to the normal controls. Under the same condition the O.D. did not show any change in 2 other patients. Purified IgG obtained from plasma of these patients had an inhibitory effect on the polymerization of human and bovine fibrinogen. IgG from normal plasma had no effect in this system. The fibrinogen of these patients had more anodic mobility than normal fibrinogen when studied by two-dimentional immunelectrophoresis. Factor XIII activity, euglobulin lysis time, and serum FDP gave results within normal range. We conclude that the prolonged thrombin time observed in some patients with LES seems to be the consequence of an abnormal fibrinogen function related to an interference by their own IgG.

The effect of antrycide of patterns of RNA synthesis in Amoeba discoides

Antrycide is an aminoquinaldine whose inhibitory action on the growth of Trypanosoma and Crithidia is not fully understood at the cellular level. The growth of Amoeba discoides in concentrations of antrycide between 0.5 and 2 microgram/ml was reduced considerably, while cells failed to divide in 4 microgram/ml. The effects on growth rate were reversible at least up until 7 days in antrycide. In order to assess the action of this synthetic drug on RNA synthesis in amoebae, the pattern of synthesis in normal cells was investigated using polyacrylamide gel electrophoresis. The profile of high molecular weight RNAs observed depended on the length of time in [3H]uridine, and was only fully developed after 66 h, when 5 peaks could be seen. The relative molecular weights of these peaks (I–V) were 2.45, 1.55, 1.13, 0.8 and 0.52 X 10(6) Daltons respectively. Those of 1.55 and 0.8 X 10(6) corresponded to ribosomal RNAs, the identity of the other peaks is unknown. After growth in 2 microgram/ml antrycide for 4 days, no high molecular weight RNA was found. Use of [14C]adenine/[3H]uridine showed that after 17 h in antrycide there was a loss of ribosomal RNA and increased levels of low molecular weight RNAs, due either to lack of synthesis or to degradation of newly synthesized material. Incorporation of [3H]leucine into hot acid-precipitable protein was inhibited in antrycide-treated cells by at least 50%. A possible explanation of the effect of antrycide on A. discoides was the inhibition of mRNA synthesis for ribosomal proteins, leading to degradation of newly synthesized rRNA. Reduced growth would continue on pre-existing ribosomes and previously synthesized long-lived mRNAs.

Soluble Fibrin Complexes During Hormonal Contraception

In a prospective study, 2o healthy women (16–35 yrs.) were treated with an oral contraceptive (o,25 mg D-norgestrel, o,o5 mg ethinyl-estradiol). Derivatives of fibrinogen and fibrin were investigated before the beginning of medication, after the 1st cycle, and after the 3rd cycle. The investigations included: determination of thrombin-clottable protein, cold precipitable protein (4°C) , ethanol precipitable protein (11,5%), ethanol gelation test, protamin sulfate test, fibrin-fibrinogen degradation products in serum, and quantitation of high-and low molecular weight derivatives of fibrinogen and fibrin by agarose gel filtration of the fresh patient plasmas.There was a significant increase of thrombin clottable protein (212 vs. 236 mg/dl, p<0,001) and an increase of ethanol precipitable protein (117 vs. 168 mg/dl, p<0,003). There was no change in the cold precipitable protein nor in the paracoagulation phenomena. Fibrin-fibrinogen degradation products were not elevated. By agarose gel filtration, a significant increase in soluble fibrin complexes (p<0,02) could be demonstrated; the highest concentration of the SFC elu-ted in the position of the dimers.The study confirms a low grade activation of the coagulation system even in healthy women taking a low dose estrogen containing oral contraceptive.