Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene.

The HindIII RFLP in the human phenylalanine hydroxylase (PAH) gene is caused by the presence of an AT-rich (70%) minisatellite region. This region contains various multiple of 30-bp tandem repeats and is located 3 kb downstream of the final exon of the gene. PCR-mediated amplification of this region from haplotyped PAH chromosomes indicates that the previously reported 4.0-kb HindIII allele contains three of these repeats, while the 4.4-kb HindIII allele contains 12 of these repeats. The 4.2-kb HindIII fragment can contain six, seven, eight, or nine copies of this repeat. These variations permit more detailed analysis of mutant haplotypes 1, 5, 6, and, possibly, others. Kindred analysis in phenylketonuria families demonstrates Mendelian segregation of these VNTR alleles, as well as associations between these alleles and certain PAH mutations. The R261Q mutation, associated with haplotype 1, is associated almost exclusively with an allele containing eight repeats; the R408W mutation, when occurring on a haplotype 1 background, may also be associated with the eight-repeat VNTR allele. Other PAH mutations associated with haplotype 1, R252W and P281L, do not appear to segregate with specific VNTR alleles. The IVS-10 mutation, when associated with haplotype 6, is associated exclusively with an allele containing seven repeats. The combined use of this VNTR system and the existing RFLP haplotype system will increase the performance of prenatal diagnostic tests based on haplotype analysis. In addition, this VNTR may prove useful in studies concerning the origins and distributions of PAH mutations in different human populations.     

Insulin-like growth factor I-like immunoreactivity in serum and tissues of the tilapia, Oreochromis mossambicus

Tilapia serum was acidified with 0.5 M HCl and then chromatographed on an octadecasily-silica column. After washing with 4% acetic acid, the column was eluted with methanol. The eluate was evaporated to dryness. The sample cross-reacted in a human insulin-like growth factor I (IGF-I) radioimmunoassay, suggesting immunochemical similarity to human IGF-I. IGF-I-like immunoreactivity was present at high levels in tilapia liver. Other tissues containing IGF-I-like immunoreactivity included the gonad, kidney, heart, spleen, brain and muscle. The serum IGF-I-like immunoreactivity was attributed to substances with a molecular weight of 9,000 and 45,000 respectively, and it was elevated after treatment with bovine growth hormone and carp pituitary extract.     

Purification and the effect of peptide N-glycosidase F on lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts.

Glucocerebrosidase was purified from human cultured dermal fibroblasts more than 2200-fold to apparent homogeneity using high performance Alkyl-Superose HR 5/5 hydrophobic interaction and Bio-Sil TSK-250 gel permeation column chromatography. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis and protein staining of the catalytically active and concentrated enzyme fractions from the gel permeation columns revealed the presence of one band of Mr 64,000. The glucocerebrosidase preparation purified to homogeneity was digested with peptide N-glycosidase F that cleaves N-linked oligosaccharide structures from glycoproteins. The molecular weight of glucocerebrosidase after digestion with peptide N-glycosidase F was reduced to Mr 57,000, suggesting that the mature enzyme is a glycoprotein and that N-linked oligosaccharide constitutes a minimum of about 10% of the total molecular weight of the polypeptide. These findings are compatible with the hypothesis that glucocerebrosidase was initially synthesized as a precursor polypeptide which was subsequently glycosylated to become the mature enzyme.     

Application of light emitting diodes as a light source to a photosynthetic culture of Chlorobium thiosulfatophilum

Summary Economical light energy supply plays a key role in determining the success of industrial application of photosynthetic microorganisms. The bacteriochlorophyll a of C. thiosulfatophilum harvests the light at 760 nm, which oxidizes toxic H₂S by photosynthesis. Light emitting diode(LED), which emits a maximum intensity at 710 nm and 60% of its maximum at 760 nm, was adopted as an alternative light source to inefficient incandescent light. With the array of the LEDs adjustable to the reactor wall 95% of the light energy was saved over the incandescent light source in comparable conditions.     

Relaxation and creep quasilinear viscoelastic models for normal articular cartilage

A quasilinear viscoelastic model was used to develop relaxation and creep forms for a constitutive law for soft tissues. Combined relaxation and cyclic test data as well as preconditioned and nonpreconditioned creep data were used to demonstrate the approach for normal bovine articular cartilage. Values for mechanical parameters in the analytical models were determined using a generalized least squares method.     

Metabolic effects of thyroid hormones at a low temperature in the snake

1. 1.|The metabolic role of the thyroid gland was studied in intact snakes, Naja naja and Ptyas korros treated with tri-iodo-thyronine (T₃) and thyroxine (T₄) and in thyroidectomized (Tx) N. naja kept at 21°C by analyzing tissue composition and glycogen phosphorylase a activity.

2. 2.|Liver weight was unaffected by thyroid hormone injection in both species but decreases in liver glycogen followed T₃ or T₄ injection, and there was an increase in liver glycogen in N. naja. These changes in liver glycogen were accompanied by a decrease in glycogen phosphorylase a activity with T₃ injection. T₃ decreased muscle glycogen in Ptyas and Tx increased it in N. naja.

3. 3.|T₃ increased % liver lipid in Ptyas but not in Naja.

4. 4.|Between species, there were differences in liver weight, blood glucose level, cholesterol level and % muscle lipid.

5. 5.|The results showed that thyroid hormones affected carbohydrate and lipid metabolism at a low temperature of 21°C, although the significance is not known.

Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene

A novel restriction fragment length polymorphism in the phenylalanine hydroxylase (PAH) locus generated by the restriction endonuclease MspI was observed in a German phenylketonuria (PKU) patient. Molecular cloning and DNA sequence analyses revealed that the MspI polymorphism was created by a T to C transition in exon 9 of the human PAH gene, which also resulted in the conversion of a leucine codon to a proline codon. The effect of the amino acid substitution was investigated by creating a corresponding mutation in a full-length human PAH cDNA by site-directed mutagenesis followed by expression analysis in cultured mammalian cells. Results demonstrate that the mutation in the gene causes the synthesis of an unstable protein in the cell corresponding to a CRM- phenotype. Together with the other mutations recently reported in the PAH gene, the data support previous biochemical and clinical observations that PKU is a heterogeneous disorder at the gene level.     

Protease Ti from Escherichia coli requires ATP hydrolysis for protein breakdown but not for hydrolysis of small peptides

Protease Ti, a new ATP-dependent protease in Escherichia coli, degrades proteins and ATP in a linked process, but these two hydrolytic functions are catalyzed by distinct components of the enzyme. To clarify the enzyme's specificity and the role of ATP, a variety of fluorogenic peptides were tested as possible substrates for protease Ti or its two components. Protease Ti rapidly hydrolyzed N-succinyl(Suc)-Leu-Tyr-amidomethylcoumarin (AMC) (Km = 1.3 mM) which is not degraded by protease La, the other ATP-dependent protease in E. coli. Protease Ti also hydrolyzed, but slowly, Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. However, it showed little or no activity against basic or other hydrophobic peptides, including ones degraded rapidly by protease La. Component P, which contains the serine-active site, by itself rapidly degrades the same peptides as the intact enzyme. Addition of component A, which contains the ATP-hydrolyzing site and is necessary for protein degradation, had little or no effect on peptide hydrolysis. N-Ethylmaleimide, which inactivates the ATPase, did not inhibit peptide hydrolysis. In addition, this peptide did not stimulate the ATPase activity of component A (unlike protein substrates). Thus, although the serine-active site on component P is unable to degrade proteins, it is fully functional against small peptides in the absence of ATP. At high concentrations, Suc-Leu-Tyr-AMC caused a complete inhibition of casein breakdown, and diisopropylfluorophosphate blocked similarly the hydrolysis of both protein and peptide substrates. Thus, both substrates seem to be hydrolyzed at the same active site on component P, and ATP hydrolysis by component A either unmasks or enlarges this proteolytic site such that large proteins can gain access to it.     

Chemical synthesis in protein engineering: total synthesis, purification and covalent structural characterization of a mitogenic protein, human transforming growth factor-alpha

Successful approaches to protein engineering required that the desired analogs be easily and rapidly obtained in sufficient quantities and purities for unambiguous structural and functional characterizations. Chemical synthesis is the method of choice for engineering small peptides. We now demonstrate that with improved methodologies and instrumentation, total chemical synthesis can be used to produce a small protein in a form suitable for engineering studies. Active human transforming growth factor-alpha (TGF-alpha), a 50 amino acid long protein with three disulfide bonds, has been synthesized and purified in multiple tens of mg amounts in less than 7 days. The purified human TGF-alpha migrated as a single band on SDS-polyacrylamide gels, ran as a single sharp major band at pI = 6.2 on isoelectric focusing gels, displayed an MW = 5546.2 (Th.5546.3) by mass spectrometry, contained three disulfide bonds and had EGF receptor binding, mitogenic and soft agar colony formation activities. The locations of disulfide bonds were found to be analogous to those found in epidermal growth factor (EGF) and in human TGF-alpha expressed in bacteria.