The appearance of alpha-crystallin in relation to cell cycle phase in the embryonic mouse lens

Specific protein synthesis in the embryonic mouse lens was studied by immunofluorescence with antisera to adult mouse lens or crystallin fractions. Positive reactions were first detected in a few cells of the lens cup 18-24 hr after contact between optic vesicle and presumptive lens ectoderm had been established. During formation of the lens vesicle a rapidly increasing fraction of cells produced crystallins. At the time of detachment of the vesicle from the surface all cells of its posterior wall showed immunofluorescence. After fiber elongation became distinct cells of the anterior epithelium began to fluoresce and shortly afterwards the entire rudiment produced crystallins. The early reactions were due entirely to the presence of alpha-crystallin. Reactions were restricted to the lens. Thus, in the mouse as in other species crystallins were detectable by immunofluorescence in vivo only after lens morphogenesis was well underway and only in the lens rudiment itself. Cells first synthesizing crystallins always had an elongated shape and their nuclei were in a basal position. A few hours later mitotic cells displayed fluorescence. Taking into account earlier found relations between cell morphology and cell cycle phase, this indicates that alpha-crystallin is first demonstrable in the S-or early G-2 phase of the cell cycle, and that the start of its synthesis does not preclude continued cell replication. It is interesting that the cellular location, cell cycle phase, and developmental stage, in which crystallins first appear, are comparable in mouse and chick embryo. Yet, entirely different proteins are involved: alpha-crystallin in the first, delta-crystallin in the latter. Implications of this for our understanding of lens induction are discussed.     

Problems encountered in measuring erythrocyte pyrimidine 5'-nucleotidase activity

An improved method of two-dimensional electrophoresis that allows unequilibrated first-dimension gels to be loaded and electrophoresed on second-dimension gels twice the length used in the O'Farrell technique has been developed. Normally, the electrophoresis of unequilibrated first-dimension gels on long second-dimension gels with the resolving gel set at pH 8.8 results in poor resolution of low-molecular-weight proteins. Adjusting the pH of the resolving gel to pH 8.3 maintains the low-molecular-weight proteins in a stacked configuration during their migration through the length of the 10% acrylamide gel. Utilization of a 10 to 20% exponential polyacrylamide gradient in a resolving gel set at pH 8.3 separates these low-molecular-weight proteins with excellent resolution. Electrophoretic resolution of protein spots in resolving gels set at pH 8.8 is not as sharp as in gels set at pH 8.3, and resolution progressively deteriorates in gels set at higher pH values.     

The sites of synthesis and the subsequent migration of newly synthesized protein in retina

Radioautographs of rabbit retinas fixed immediately after a 1 or 2 min exposure in vitro to ³H leucine revealed high rates of protein synthesis in receptor cell inner segments, perikarya of ganglion cells, and cells of the inner nuclear layer. If these brieflly labelled retinas were returned to unlabelled medium for periods of up to 6 hr, the radioautographs revealed a progressive dispersion of the labelled proteins from their sites of synthesis. This was largely completed by $\text{1}\text{1}\text{2}$ hr and appeared, in one instance at least, to involve processes other than simple diffusion. Superimposed on the dispersive phenomenon was a process of concentration of the newly formed proteins at two sites quite distant from their synthesis, that was apparent after $\text{1}\text{1}\text{2}$hr. One of these sites was the receptor cell outer segments, as has been previously described, the other was the outer plexiform layer.     

Simplified radioimmunoassay for aldosterone using antisera to aldosterone-γ-lactone

A simplified, non-chromatographic method for aldosterone-γ-lactone (2) radioimmunoassay is described, using a high titer aldosterone-γ-lactone antibody. This method takes six working hours for completion; the blanks are below the sensitivity of the assay (0–10 pg), and the intra and interassay coefficient of variation is 4.9% and 8.2% respectively. There is no significant variation with different plasma volumes assayed and the plot of added and assayed aldosterone gives a slope of 0.983. The negligible cross reactivity with other steroids and lactones eliminates the problem of interference by these substances.     

The occurrence of fluoride stimulated membrane phosphoprotein phosphatase

Membrane preparations from rabbit peritoneal granulocytes and dog blood platelets possess an active phosphoprotein phosphatase. The enzyme is stimulated by fluoride and to a lesser extent by prostaglandin E₁ (PGE₁). It dephosphorylates ³²P labeled, catalytically active phosphoglucomutase (PGM) and labeled endogenous membranes to yield, in both cases, inorganic phosphate. It is inactive towards denatured PGM, denatured endogenous membranes and thymus histone labeled with ³²P.     

Thromboxane and pulmonary hypertension following E. coli endotoxin infusion in sheep: Effect of an imidazole derivative

We assessed the effect of a specific thromboxane synthetase inhibitor (an imidazole derivative) on pulmonary hemodynamics and the concentrations of TxB₂ (TxA₂), 6-keto-PGF_1α (PGI₂), and PGF_2α in pulmonary lymph and transpulmonary blood samples following intravenous administration of E. coli endotoxin (1 μg/kg) in sheep. In control animals the rise in pulmonary artery pressure correlated with increases in plasma and lymph TxB₂ concentrations and large transpulmonary concentration gradients of this metabolite were measured. In imidazle treated animals both pulmonary hypertension as well as increases in plasma and lymph TxB₂ concentrations were substantially reduced. In contrast, peak concentrations of 6-keto-PGF_1α (PGI₂) and PGF_2α were severalfold higher than those measured in control animals. This suggests a shunting of endoperoxide metabolism towards prostacyclin and primary prostaglandins and documents the specificity of the thromboxane synthetase inhibitor. Out study provides evidence that endotoxin-induced pulmonary hypertension is mediated by pulmonary synthesis of TxA₂.     

On the biochemical similarities of ascorbic acid and interferon

The anti-viral effects of ascorbic acid and interferon are proposed to result from the peroxy radical mediated biosynthesis of prostaglandins and from the direct attack of free radical species on unsaturated lipids in the cell membrane. These processes are proposed to lead to changes in fluidity of the host cell membrane which inhibit the fusogenicity of the attacking virus.     

Effect of dietary proteins and carbohydrates on urinary and sympathoadrenal catecholamines

We previously observed that administration of tyrosine to rats or humans elevated urinary dopamine, norepinephrine and epinephrine levels. The present studies examine the effects on these urinary catecholamines of varying the ratio of protein to carbohydrate in the diets.Rats consumed diets containing 0, 18 or 40% protein (76, 58 and 36% carbohydrate respectively) for 8 days. The stress of consuming the protein-free food was associated with a 16% weight reduction, and with significantly lower serum, heart and brain tyrosine levels than those noted in rats eating the 18 or 40% protein diets. Absence of protein from the diet also decreased urinary levels of dopamine and DOPA but increased urinary norepinephrine and epinephrine, probably by increasing sympathoadrenal discharge; it also increased the excretion of DOPA in animals pretreated with carbidopa, a DOPA decarboxylase inhibitor. Carbidopa administration decreased urinary dopamine, norepinephrine and epinephrine as expected; however, among carbidopa-treated rats urinary norepinephrine and epinephrine concentrations were highest for animals consuming the protein-free diet, again suggesting enhanced release of stored catecholamines from sympathoadrenal cells. The changes in urinary catecholamines observed in animals eating the protein-free diet were similar to those seen in rats fasted for 5 days: dopamine levels fell sharply while norepinephrine and epinephrine increased.These data indicate that the effects of varying dietary protein and carbohydrate contents on dopamine secretion from peripheral structures differ from its effects on structures secreting the other two catecholamines. Protein consumption increases dopamine synthesis and release probably by making more of its precursor, tyrosine, available to peripheral dopamine-producing cells; it decreases urinary norepinephrine and epinephrine compared with that seen in protein-deprived animals, probably by diminishing the firing of sympathetic neurons and adrenal chromaffin cells.     

Purification of a brain-specific astroglial protein by immunoaffinity chromatography

An immunoaffinity chromatography procedure for the isolation of bovine glial fibrillary acidic (GFA) protein is described. Degraded GFA protein isolated by hydroxyapatite chromatography from human spinal cord was used to prepare the antiserum. The immunoglogulin G fraction of the antiserum was covalently linked to CNBr-activated Sepharose, and columns of the immuno-affinity gel were used to adsorb bovine GFA protein from brain extracts. Elution was accomplished with a solution of 1 m acetic acid, 5 m urea, 0.8 m sodium chloride, pH 2.5. The yield of about 0.5 mg of highly purified protein/g of cerebral white matter could be increased to 1.5 mg/g of tissue by lowering the ionic strength of the extracting buffer from 50 mm to 1 mm sodium phosphate. Isolation in the presence of EDTA prevented the formation of an oxidation product migrating as a dimer of the monomeric species on SDS-polyacrylamide gel electrophoresis.     

Scoyenia Escape Burrows in Fluvial Pebbly Sand: Upper Triassic Sugarloaf Arkose,Deerfield Rift Basin,Massachusetts, USA

Late Triassic larvae of an insect, probably a beetle, moved diagonally upwards though fluvial pebbly sand along a thin mud layer, constructing Scoyenia burrows in the Sugarloaf Arkose, Deerfield rift basin, Massachusetts. They may have been escaping a temporary rise of the water table in the monsoonal dry season.