The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Pale and dark bipolar cells in the chicken retina

Ultrastructurally, two different bipolar cell types can be distinguished in the retina of the chick embryo: one pale or electron-lucent and the other dark or electron-dense. The different electron density was seen over the whole cell, from its enclave in the outer limiting membrane to its termination in the inner plexiform layer. These observations prompted us to study the content and cytoplasmic variations of both cell types. The pale bipolar cell has a higher vacuole, vesicle and endoplasmic reticulum content and a lower number of microtubules and glycogen than the dark bipolar cell. The presence of these two cell types is probably due to a characteristic physiologic state, and the difference between the pale and dark bipolar cells can be attributed to the diverse number of gap unions which they establish with A II amacrine cells.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Fluorescence properties of catecholamines in isolated storage organelles of adrenal medulla

THe quantum yield, the life time and the degree of polarization of the fluorescence of intact chromaffin granules isolated from bovine adrenal medulla were compared to those of catecholamines solutions and catecholamine/ATP mixtures. Rising concentrations of catecholamines in aqueous solutions exhibited increasing quenching and decreasing life times indicating that the quenching was collision induced. Similar effects occurred in mixtures of catecholamines with ATP and Ca²⁺ showing that the nucleotide did not remarkedly hinder the mobility of the catechol group. In suspensions of whole granules stron quenching and shortening of life time was observed compared with solutions of disrupted granules. Fluorescence yield and life time were decreased by about the same factor suggesting that storage of the amines was not correlated with a major immobilization of the catechol group. The degree of polarization of intact granules was higher than that of solutions of catecholamines alone, but similar to catecholamine/ATP mixtures with concentrations corresponding to those found in the granules. This indicates an interaction of catecholamines with ATP in the granules. The results are in agreement with a storage model for catecholamines in the chromaffin granules of adrenal medulla in which catecholamines are bound to ATP, but in a non-rigid way.     

Two modes of cell migration in the ventral horn of the spinal cord in the chick embryo. A Golgi study

The migration process of the ventral horn in chick embryo spinal cord cells has been studied between 2.5 and 5 days of incubation (HH-17, HH-26), using the Golgi technique. Two different migratory modes are observed. Type I--Migration by nucleus translocation. Most of the ventral horn motor neurons migrate by nucleus translocation within the peripheral cylinder of the cytoplasm (migration by nucleus translocation). Type II--Free migration cells. Other cells migrate disconnected from both limiting surfaces (ventricular and pial). On the basis of shape and migratory behaviour they have been identified as smooth cells and multipodial cells.     

Determination of fatty acid and triacylglycerol composition of human very-low-density lipoproteins

The fatty acid composition of human very-low-density lipoproteins (VLDL) was studied in a population from western Andalusia with a diet in which the fat content came mainly from olive oil. The lipid composition of VLDL, including the fatty acid composition of the phospholipids and triacylglycerols, was examined by capillary gas chromatography. Twenty-five peaks were resolved, ranging in chain length from 14 to 24 carbon atoms, including geometric and positional isomers. The major fatty acids present in phospholipids were 16:0, 18:0, 18:1(n − 9) and 18:2(n − 6), and in triacylglycerols were 18:1(n − 9), 16:0 and 18:2(n − 6). The major triacylglycerol was POO, followed by PLO and OOO. MLP, PPS and LLL were absent. The presence of a large amount of OOO in this fraction demonstrates that the triacylglycerol composition of the VLDL depends on the type of diet consumed.     

Turning in MFB-lesioned rats and antagonism of neuroleptic-induced catalepsy after lisuride and LSD.

The effects of lisuride, d-lysergic acid diethyl amide (LSD) and apomorphine were studied in rats with unilateral destruction of nigro-striatal nerve terminals either with 6-hydroxydopamine (6-OHDA) or 5, 6-dihydroxytryptamine (5,6-DHT). Lisuride at the dose of 50 μg kg^?1 i.p. induced contralateral turning for more than 4 hours while the circling induced by LSD (200 μg kg^?1) and apomorphine (1 mg kg^?1) persisted for only one hour. Lisuride, a compound stimulating both dopamine (DA) and 5-hydroxytryptamine (5-HT) receptors induced a more intense turning in 6-OHDA than in 5,6-DHT lesioned rats. This might indicate a modulation of 5-HT on rotational behavior. Haloperidol (1 mg kg^?1 i.p.) antagonized both lisuride- and LSD-induced turning. LSD, and much more persistently lisuride, counteracted the prochlorperazine-induced catalepsy. These findings correlate with the biochemical data indicating that lisuride is a very potent agonist at central dopaminergic receptors.     

Extracellular Concentrations of Dopamine and Metabolites in the Rat Caudate After Oral Administration of a Novel Catechol-O-Methyltransferase Inhibitor Ro 40–7592

The effect of the systemic administration of a novel, orally active, catechol-O-methyltransferase (COMT) inhibitor, Ro 40-7592, on the in vivo extracellular concentrations of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), was studied by transcerebral microdialysis in the dorsal caudate of freely moving rats. Ro 40-7592 (at doses of 3.0, 7.5, and 30 mg/kg p.o.) elicited a marked and long-lasting reduction of HVA, and at doses of 7.5 and 30 mg/kg, an increase of DOPAC output, but it failed to increase DA output. The administration of L-beta-3,4-dihydroxyphenylalanine (L-DOPA, 20 and 50 mg/kg p.o.) with a DOPA decarboxylase inhibitor (benserazide) increased both HVA and DOPAC output, but failed to modify significantly extracellular DA concentrations in dialysates; in contrast, combined administration of L-DOPA+benserazide with Ro 40-7592 (30 mg/kg p.o.) resulted in a significant increase in DA output. Ro 40-7592 prevented the L-DOPA-induced increase in HVA output and markedly potentiated the increase in DOPAC output. To investigate to what extent the increase in extracellular DA concentrations was related to an exocitotic release, tetrodotoxin (TTX) sensitivity was tested. Addition of TTX to Ringer, although abolishing DA output in the absence of L-DOPA, partially reduced it in the presence of L-DOPA+Ro 40-7592 and even more so after L-DOPA without the COMT inhibitor. The results of the present study suggest that metabolism through COMT regulates extracellular concentrations of DA formed from exogenously administered L-DOPA but not of endogenous DA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.