Analysis of the nuclear envelope polypeptides by isoelectric focusing and electrophoresis

The more insoluble polypeptides of the avian erythrocyte nuclear envelope have been characterized by a two-dimensional electrophoretic procedure. Most of the polypeptides occur in two classes with isoelectric points of approximately 6.4 and 5.7 respectively. The more acidic class contains two polypeptides, P71 and one which contributes to an electrophoretic band previously identified as P55. The more basic class includes P75, P68, P61 and two or more polypeptides from the P55 band. There are four to six isoelectric point variants of each polypeptide in the more basic class, and the relative stain intensities for the variants are similar for the different polypeptides. These similarities in ionic properties suggest a chemical relationship between the polypeptides. These results are discussed in relation to the in vitro conversion of P75 to polypeptides of the same molecular weight as P68, P61 and P55.     

Sucrose dilution of glycerol from mouse embryos frozen rapidly in liquid nitrogen vapour

The toxic effects of sucrose and the conditions of in-straw glycerol removal after freezing and thawing were studied using Day-3 mouse embryos. At 20 degrees C, exposure to less than or equal to 1.0 M-sucrose for periods up to 30 min had no adverse effects on freshly collected embryos. At 25 and 36 degrees C, however, greater than or equal to 1.0 M-sucrose significantly reduced the developmental potential (P less than 0.001). In the freezing experiments the embryos were placed in 0.5 ml straws containing 40 microliters freezing medium separated by an air bubble from 440 microliters sucrose solution. The straws were frozen rapidly in the vapour about 1 cm above the surface of liquid nitrogen. The post-thaw viability was substantially better after sucrose dilution at 20 degrees C than at 36 degrees C. Mixing the freezing medium with the sucrose diluent immediately after thawing further improved the rate of survival relative to mixing just before freezing (P less than 0.001). The best survival was obtained when the freezing medium contained 3.0 M-glycerol + 0.25 M-sucrose; it was mixed with the diluent after thawing and the glycerol was removed at 20 degrees C. Under such conditions the sucrose concentration in the diluent had no significant effect on the rate of development (0.5 M, 69%; 1.0 M, 73%; 1.5 M, 64%). The results show that during sucrose dilution the temperature should be strictly controlled and suggest that intracellular and extracellular concentrations of glycerol are important in the cryoprotection of embryos.     

On the mechanism of sequence iron-hematein stains

Summary As a prerequisite for the histochemical study of sequence iron-hematoxylin stains the iron alum-acidified hematein procedure was developed which does not require differentiation.Histochemical blocking and extraction procedures demonstrated that carboxyl and hydroxyl groups are essential for the binding of cationic iron.The iron alum-Prussian blue reaction colored collagen, reticulum fibers and basement membranes more intensely than muscle fibers. Treatment of tissue sections mordanted in iron alum with the acidified hematein solvent resulted in practically complete removal of iron from all tissue structures. It must therefore be concluded that the selective staining of muscle fibers, terminal bars and related structures with sequence iron-hematein stains is not due to high affinity of iron for these tissue components.Observations by R. and M. Heidenhain on sequence hematoxylin-potassium dichromate and hematoxylin-alum stains and data from modern textile chemistry indicate that the staining patterns obtained with metal-hematein sequence stains are determined by the affinity of the hematein moiety for certain tissue structures.     

The genetic analysis of a large transposing element of Drosophila melanogaster

A member of Ising's family of large transposing elements (TEs) has inserted into, or very near, the crinkled (ck, 2–50) locus. This TE (TE36) carries functional alleles of both the white and roughest loci, and causes a hypomorphic mutation of ck. The TE is visible in polytene chromosomes as a two-banded insertion between 35B9 and 35C1. These bands show homology to foldback (FB) elements by in situ hybridization. All spontaneous losses of TE36 remain mutant for ck and retain sequences homologous to FB at the site of TE's insertion. TE36 carries only one functional copy of w ⁺, by the criterion that z w, TE36/ + flies are wild-type for eye color but z w; TE36/TE36 flies are zeste. This white⁺ gene is dosage compensated since w/Y; TE36/+ males have twice as much eye pigment as w/w; TE36/ + females. A form of the TE that has four polytene chromosome bands and expresses twice as much pigment as TE36 has been recovered. However, its white genes are not suppressed by zeste.     

Silver impregnation methods for reticulum fibers and reticulin: A re-investigation of their origins and specifity

Summary Maresch (1905) introduced Bielschowsky's silver impregnation technic for neurofibrils as a stain for reticulum fibers, but emphasized the nonspecifity of such procedures. This lack of specifity has been confirmed repeatedly. Yet, since the 1920's the definition of reticulin and studies of its distribution were based solely on silver impregnation technics. The chemical mechanism and specifity of this group of stains is obscure. Application of Gomori's and Wilder's methods to human tissues showed variations of staining patterns with the fixatives and technics employed. Besides reticulum fibers, various other tissue structures, e.g. I bands of striated muscle, fibers in nervous tissues, and model substances, e.g. polysaccharides, egg white, gliadin, were also stained. Deposition of silver compounds on reticulum fibers was limited to an easily removable substance; the remaining collagen component did not bind silver. These histochemical studies indicate that silver impregnation technics for reticulum fibers have no chemical significance and cannot be considered as histochemical technics for reticulin or type III collagen.     

A radiochemical assay for N-acetyl-L-aspartate amidohydrolase (EC 3.5.1.15) and its occurrence in the tissues of the chicken

A simple and rapid radiochemical method for the determination of N-acetyl-L-aspartic acid amidohydrolase (EC 3.5.1.15) activity using ion exchange chromatography has been developed. The activity of this enzyme in the developing brain and some non-nervous tissues of the chicken has been determined. No activity of the enzyme could be detected in the brains of chick embroys prior to 14 days of gestation; activities gradually increased thereafter to adult levels which are about 60% of that found in the adult rat. In non-nervous system tissues of the adult chicken, activities varied from high levels in the kidney to low levels in heart and breast muscle. Treatment of the homogenates of the adult tissues with a detergent significantly increased the enzyme activity, suggesting that a portion of the enzyme is membrane bound.     

Effects of synthetic ovine CRF on ACTH, cortisol and blood pressure in sheep

Intravenously administered synthetic ovine CRF at doses of 0.1, 1.0 and 10.0 micrograms/kg increased plasma ACTH and cortisol concentrations in a dose-dependent fashion in unanesthetized sheep. In two unanesthetized sheep, aortic blood pressure remained relatively unaffected after the intravenous administration of CRF at 5 and 20 micrograms/kg. These results suggest that peripherally administered ovine synthetic CRF specifically stimulates the sheep pituitary-adrenal axis. Unlike other species receiving intravenous synthetic ovine CRF, sheep did not show hypotensive effects.     

An unusual phenotypic expression of Hb-Leiden

After a boy of Mexican-American descent became jaundiced during treatment of a serious urinary tract infection with an oxidant drug, an extensive hematological examination was made. The important finding was the presence of Hb-Leiden to the extent of less than 3% or about a tenth of the usual percentage. Although inclusion bodies are present in the erythrocytes at all times, his hematological parameters have remained normal. The genetic basis for the unusually small amount of Hb-Leiden in the propositus may be due to the Hb-Leiden gene in an anti-Lepore configuration, that is, and Hb-Leiden gene in cis to the normal β and δ genes.     

Excitotoxic Brain Injury Suppresses Striatal High-Affinity Glutamate Uptake in Perinatal Rats

In immature rodent brain, the glutamate receptor agonist N-methyl-D-aspartate (NMDA) is a potent neurotoxin. In postnatal day (PND)-7 rats, intrastriatal injection of 25 nmol of NMDA results in extensive ipsilateral forebrain injury. In this study, we examined alterations in high-affinity [3H]glutamate uptake (HAGU) in NMDA-lesioned striatum. HAGU was assayed in synaptosomes, prepared from lesioned striatum, the corresponding contralateral striatum, or unlesioned controls. Twenty-four hours after NMDA injection (25 nmol), HAGU declined 44 +/- 8% in lesioned tissue, compared with the contralateral striatum (mean +/- SEM, n = 6 assays, p less than 0.006, paired t test). Doses of 5-25 nmol of NMDA resulted in increasing suppression of HAGU (5 nmol, n = 3; 12.5 nmol, n = 3; and 25 nmol, n = 5 assays; p less than 0.01, regression analysis). The temporal evolution of HAGU suppression was biphasic. There was an early transient suppression of HAGU (-28 +/- 4% at 1 h; p less than 0.03, analysis of variance, comparing changes at 0.5, 1, 2, and 3 h after lesioning); 1 or 5 days postinjury there was sustained loss of HAGU (at 5 days, -56 +/- 11%, n = 3, p less than 0.03, paired t test, lesioned versus contralateral striata).(ABSTRACT TRUNCATED AT 250 WORDS)     

A potent and selective photoaffinity probe for the anti-estrogen binding site of rat liver

The anti-estrogen binding site (ABS) is an apparently ubiquitous component of cells that has been shown to be intimately linked with the antiproliferative effects of certain antiestrogenic compounds, like tamoxifen, which is currently used for the treatment of breast cancer. However, the identification and in vitro study of this novel protein has been hampered to date by a lack of convenient probes that will efficiently label the molecule in nonpurified preparations. Thus, using a selective ABS ligand (4-benzylphenoxy-N-ethylmorpholine, MBPE) as starting material, we synthesized a photosensitive azido derivative, [(2-azido-4-benzyl)phenoxy]N-ethylmorpholine (azido-MBPE) that can be prepared in a tritiated form. Azido-MBPE has a high affinity for ABS (Kd = 3 nM), identical to that of tamoxifen, and covalently labels 5 and 12% of membrane-bound and detergent-solubilized ABS, respectively. Its incorporation is selectively and competitively inhibited by other ABS ligands (tamoxifen greater than nitromifen greater than hydroxytamoxifen). [3H]Azido-MBPE potently photolabels either membrane-bound or detergent-solubilized ABS as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions revealing specific photoincorporation in a protein band of Mr = 40,000. This molecular weight is approximately two times lower than what we observed previously for ABS preparations studied under nondenaturing conditions and postlabeled with [3H]tamoxifen (Mr = 80,000-110,000). In chromatofocusing experiments with photolabeled ABS, a single specifically labeled protein fraction migrating with a pI of 6.4 was found to exhibit a Mr of 40,000 when subsequently electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. These results indicate that [3H]azido-MBPE is a specific high affinity probe of ABS that will prove useful in the ultimate identification of this protein.