Evidence for polarizing zone in the limb buds of Xenopus laevis

To test for the presence of polarizing mesoderm in an amphibian, Xenopus laevis hindlimb bud tips were rotated 180° on the proximodistal axis and returned to the stump. Supernumerary outgrowths were induced in the preaxial stump and preaxial tip tissues, and the most postaxial digit always formed next to the grafted postaxial tissue. The occurrence of polarized supernumerary outgrowths indicated that the posterior limb border contained a polarizing zone. When the limb tip was cut at varying known lengths from the body wall, rotated, and grafted to the limb stump, the incidence of twinning along the proximodistal axis permitted insight into the distribution of the polarizing zone along the posterior border. The location of polarizing tissues was found to be similar to that in the chick wing bud at comparable stages. To confirm the posterior border stump influence on the rotated preaxial limb tip tissues, 180° tip rotations were made at the proximodistal level with the highest incidence of twinning. In these cases, the adjacent stump posterior border tissues (polarizing zone) were removed, leaving a substantial amount of the deeper postaxial stump tissue, however. The frequency of twinning from tip tissues was greatly reduced in these larvae compared to those with rotated limb tips on intact stumps. Cytological examination of supernumerary outgrowths resulting from grafts of two-nucleolate tips onto one-nucleolate stumps confirmed the preaxial source of the supernumerary outgrowths.     

Liquid chromatographic demonstration of catecholamine release in fish heart.

High-pressure liquid chromatography (HPLC) was used to assay for catecholamines released into the heart of the goldfish (Carassius auratus L.) upon stimulation of vagal nerve fibers. Stimulation induced the release of norepinephrine, epinephrine and dopamine into the lumen of the perfused, isolated heart. The results suggest that heart activity in teleosts is directly modulated by functional adrenergic, cardioacceleratory nerve fibers.     

Ammonia excretion in the seawater blue crab (Callinectes sapidus) occurs by diffusion, and not Na+/NH4+ exchange

Summary A series of experiments was conducted to investigate whether ammonia is excreted across the seawater-acclimated blue crab's gills as ionized NH ₄ ⁺ or as the free base, NH₃. The net excretion rate of ammonia was not changed by transfer of the crabs to reduced (150 mM) Na⁺ solutions, by transfer to Na⁺- and K⁺-free artificial sea water, or by the sodium transport inhibitor amiloride. Ammonia excretion, therefore, does not appear to be linked to Na⁺ uptake in these animals, and appears to take place by passive diffusion. Since ammonia could diffuse either as NH ₄ ⁺ or NH₃, we examined two other kinds of evidence. The trans-epithelial potential was measured in sea water and the various artificial media. In spite of a 10 mV more negative potential in Na⁺-, K⁺-free medium, the ammonia excretion was not reduced. Also, in alkalinized seawater in which the partial pressure gradient of NH₃ was reduced, but the concentration gradient of NH ₄ ⁺ increased, ammonia excretion was reduced by about 70%. These results are consistent with the conclusion that ammonia excretion takes place by diffusion of the free base, NH₃.Abbreviations SW sea water - ASW artificial sea water - t.e.p. transepithelial potential The University of Texas Marine Science Institute Contribution No. 461Supported by NSF Grant PCM77-24358     

The metabolism of benzyl isothiocyanate and its cysteine conjugate.

1. The corresponding cysteine conjugate was formed when the GSH (reduced glutathione) or cysteinylglycine conjugates of benzyl isothiocyanate were incubated with rat liver or kidney homogenates. When the cysteine conjugate of benzyl isothiocyanate was similarly incubated in the presence of acetyl-CoA, the corresponding N-acetylcysteine conjugate (mercapturic acid) was formed. 2. The non-enzymic reaction of GSH with benzyl isothiocyanate was rapid and was catalysed by rat liver cytosol. 3. The mercapturic acid was excreted in the urine of rats dosed with benzyl isothiocyanate or its GSH, cysteinyl-glycine or cysteine conjugate, and was isolated as the dicyclohexylamine salt. 4. An oral dose of the cysteine conjugate of [14C]benzyl isothiocyanate was rapidly absorbed and excreted by rats and dogs. After 3 days, rats had excreted a mean of 92.4 and 5.6% of the dose in the urine and faeces respectively, and dogs had excreted a mean of 86.3 and 13.2% respectively. 5. After an oral dose of the cystein conjugate of [C]benzyl isothiocyanate, the major 14C-labelled metabolite in rat urine was the corresponding mercapturic acid (62% of the dose), whereas in dog urine it was hippuric acid (40% of the dose). 5. Mercapturic acid biosynthesis may be an important route of metabolism of certain isothiocyanates in some mammalian species.     

Analysis of chromosomal integration and deletions of yeast plasmids.

Plasmid DNAs from six strains of Saccharomyces cerevisiae were compared. Three different plasmids were found, designated Scp 1, Scp 2 and Scp 3, with monomer lengths of 6.19, 6.06 and 5.97 kilobases as referenced to sequenced phiX174 DNA. DNA from each of the plasmids was inserted into a lambda vector DNA. Hybrid phage containing inserted DNA of the desired size were enriched by genetic selection and their DNAs analysed by rapid techniques. All three plasmids share the same organization, two unique sequences separated by two inverted repeats, and share basically the same DNA sequences. Scp 2 and Scp 3 differ from Scp 1 by missing a unique HpaI site and by having small overlapping deletions in the same region. The HpaI site in Scp 1 is, therefore, in a nonessential region and suitable for insertion of foreign DNA in the potential use of the yeast plasmid as a vector. Hybridization of labelled cloned plasmid DNA to restriction fragments of linear yeast DNA separated on agarose gels showed that the plasmid DNA was not stably integrated into the yeast chromosomal DNA.     

Relationship between functional castration and alpha-fetoprotein produced by hepatoma-bearing female rats.

In rats bearing Morris hepatoma No. 7777, serum levels of alpha fetoprotein (AFP) increased as tumour size increased. Hepatoma-bearing females remained in dioestrus once the tumour exceeded a cross-sectional area of 12 cm2. The following changes were seen in tumour-bearing rats compared to control animals: uterine wet weight was significantly decreased, the uterine epithelial mucosa was lower, castration cells developed in the anterior pituitary, and there was a 3- to 23-fold elevation in serum oestrogen levels. We conclude that circulating oestrogen is bound to AFP in hepatoma-bearing female rats and that in this bound state it is unable to exert its normal physiological actions.     

Tandem integration of complete and defective SV40 genomes in mouse-human somatic cell hybrids.

We have analyzed the arrangement of SV40 DNA sequences integrated in human chromosome 7 in two lines of mouse-human somatic cell hybrids: one containing only one human chromosome 7 per cell and the other containing an average of about three. We found that the integration site differs in both the viral and host sequences in the two clones. However, the sites of integration into the several copies of human chromosome 7 of one clone are identical. Each chromosome 7 in both clones carries approximately six viral genomes tandemly linked. Some of these genomes lack about 20% of the DNA from the late region, including the Eco RI site.     

Prevalence of severe growth hormone deficiency.

Four hundred and forty-nine short children, who were all over 2-5 standard deviations below the mean height for age, were identified by screening the heights of 48 221 6- to 9-year-old children in three Scottish cities. Most were screened for growth hormone deficiency (GHD). The prevalence of severe GHD in this sample may have been as high as 1 in 4018, much higher than reported. The findings suggest that present referral patterns may account for the delayed or missed diagnosis of the condition in girls or children with less severe short stature.