L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996     

Influence of thyroid hormones on the human ATP synthase β-subunit gene promoter

The action of thyroid hormones on the expression of the mitochondrial ATP synthase -subunit gene (ATPsyn) is controversial. We detected a binding site for the thyroid hormone receptor between-366 and-380 in the human ATPsyn gene by DNase I footprint analysis and band-shift assays. However, expression vectors in which the chloramphenicol acetyl transferase (CAT) reporter gene is driven by the 5 upstream region of ATPsyn gene were unresponsive to T₃ when transiently transfected to HepG2 or GH4C1 cells. CAT constructs driven by the rat phosphoenolpyruvate carboxykinase (PEPCK) or the growth hormone (GH) promoters were stimulated several fold by T₃ in parallel experiments. It is proposed that the biological effects of thyroid hormones on the ATPsyn expression occur through indirect mechanisms.     

The use of double fluorescence in situ hybridization to physically map the positions of 5S rDNA genes in relation to the chromosomal location of 18S-5.8S-26S rDNA and a C genome specific DNA sequence in the genus Avena.

A physical map of the locations of the 5S rDNA genes and their relative positions with respect to 18S-5.8S-26S rDNA genes and a C genome specific repetitive DNA sequence was produced for the chromosomes of diploid, tetraploid, and hexaploid oat species using in situ hybridization. The A genome diploid species showed two pairs of rDNA loci and two pairs of 5S loci located on both arms of one pair of satellited chromosomes. The C genome diploid species showed two major pairs and one minor pair of rDNA loci. One pair of subtelocentric chromosomes carried rDNA and 5S loci physically separated on the long arm. The tetraploid species (AACC genomes) arising from these diploid ancestors showed two pairs of rDNA loci and three pairs of 5S loci. Two pairs of rDNA loci and 2 pairs of 5S loci were arranged as in the A genome diploid species. The third pair of 5S loci was located on one pair of A-C translocated chromosomes using simultaneous in situ hybridization with 5S rDNA genes and a C genome specific repetitive DNA sequence. The hexaploid species (AACCDD genomes) showed three pairs of rDNA loci and six pairs of 5S loci. One pair of 5S loci was located on each of two pairs of C-A/D translocated chromosomes. Comparative studies of the physical arrangement of rDNA and 5S loci in polyploid oats and the putative A and C genome progenitor species suggests that A genome diploid species could be the donor of both A and D genomes of polyploid oats. Key words : oats, 5S rDNA genes, 18S-5.8S-26S rDNA genes, C genome specific repetitive DNA sequence, in situ hybridization, genome evolution.     

Molecular characterization of a fourth isoform of the catalytic subunit of protein phosphatase 2A from Arabidopsis thaliana

We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5 terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.     

Southern elephant seal,Mirounga leonina: composition of milk during lactation

An analysis of milk constituents during various stages of lactation in the southern elephant seal Mirounga leonina was carried out. Forty-six milk samples were taken from 30 females throughout lactation during 1985, 1987, 1990 and 1991 on Stranger Point, King George Island, South Shetland Islands. Total nitrogen (TN), non-protein nitrogen (NPN), sugar, fat, ash and water were measured, and from some of these data true protein and energy content were calculated. The results showed a high degree of variation in water and fat concentrations among samples at different stages of lactation. During the first 20 days the fat content of milk increased from about 12 to approximately 52%, while water content fell from 70 to 33%. The composition of milk changes rapidly during the first days post-partum. Protein, minerals and sugar appear to remain stable after the fourth day of lactation. Milk samples contain significant levels of sugars; thin layer chromatography indicates the presence of lactose and glucose together with other unidentified components. There is evidence of a striking change in composition of the milk in the later part of lactation; the progressive increase in the fat:water ratio is abruptly reversed just prior to weaning.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: X. Pregnancy diagnosis in Perissodactyla

Enzymeimmunoassays (EIAs) for estrone conjugates (EC), pregnanediol-3-glucuronide (PDG), and C-19 and C-21 progesterone metabolites (C-19/C-21) were used to analyze urine samples from four nondomestic equid species, four tapir species, and two rhinoceros species in an attempt to identify if these assays could be used for diagnosing and monitoring pregnancy. The same urine samples were also analyzed for the presence of equine chorionic gonadotropin (eCG) activity, using a field dipstick test and a radioimmunoassay (RIA). The EC EIA was validated for three equid species and the Malayan tapir. Neither the PDG nor the C-19/C-21 EIAs were validated in any species evaluated. In equid species, the EC EIA demonstrated a specificity (the percentage of nonpregnant samples identified correctly) of 100% and a sensitivity (the percentage of pregnant samples identified correctly) of ≥ 88%. With the exception of the Grevy's zebra, the C-19/C-21 EIA showed a similar accuracy in identifying pregnant and nonpregnant equids. The PDG EIA was not sufficiently accurate to merit its use in equids or tapirs for pregnancy diagnosis. From the data collected, it appears analysis of a single urine by both the EC EIA and the C-19/C-21 EIA would be the best method of pregnancy detection during the last 2 trimesters of gestation, in equid species. In tapirs, the C-19/C-21 EIA was slightly more accurate for pregnancy diagnosis than the EC EIA. The C-19/C-21 EIA had a specificity of 93%, but a sensitivity of only 73% in tapir species. None of the EIAs evaluated demonstrated a sufficient specificity or sensitivity to be useful, as presently performed, for pregnancy diagnosis from a single sample in the black rhinoceros. The eCG dipstick used in this study did not prove a sufficiently reliable test for routine pregnancy in nondomestic equids. The eCG RIA results in the Przewalski's horses and the Hartman's mountain zebra were positive early in gestation, and indicate that gonadotropin analysis may be useful for pregnancy detection in these species. Only very low amounts of eCG activity was measured by the eCG RIA in the tapir and rhinoceros urine samples. © 1994 Wiley-Liss, Inc.     

Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen. A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79–80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.     

A structural and evolutionary analysis of a dispersed repetitive sequence

A family of dispersed repetitive sequences (Hch1) which is present in the genome of the wild barley Hordeum chilense was studied in detail. Hch1 sequences are found both as part of short tandem arrays and dispersed throughout the H. chilense chromosomes. Subcloning of sections of the sequence reveals that it is composed of unrelated classes of sequences which can also be found separately in other genomic locations. Analysis of these sequences in the genomes of wheat and two other wild barley species strongly suggests that specific amplifications and arrangements of the repeated sequences have taken place during speciation. Nucleotide sequence analysis fails to detect, in their entirity, the features shown by plant transposons.     

CD studies of peptides that mimic the four helicoidal sequences of the uteroglobin monomer

Summary Short peptides spanning the helicoidal sequences of the uteroglobin monomer (crystal forms P2₁ and C222₁) were synthesized and studied by circular dichroism spectroscopy. None of them showed any secondary structure in the absence of HFIP. However, most peptides achieved a helical conformation when this structuring agent was used, with the exception of the analogue corresponding to the helicoidal fragment 19–24 (helix II, crystal P2₁). These results indicate that other factors, such as interchain interactions, have to contribute to helix stabilization in the molecule. On the other hand, while peptides corresponding to N- and C-terminal fragments that contain the first and fourth helices of the monomer, respectively (1–14 and 48–70) achieved a -like structure when 10–15% of HFIP was used, this behaviour was not observed when TFE was used. Moreover, substitution of cysteine by -aminobutyric acid at position 3 increased both the helicity of fragment 1–14 and its ability to adopt a -like structure, but the opposite effect was observed for fragment 48–70 when -aminobutyric acid was introduced at position 69. These results indicate that this part of the protein might be sensitive to the chemical environment it is exposed to and that the two cysteine residues at positions 3 and 69 of the monomer could play a different role in the folding process.