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Biomedical subjects

R Lahti

Publications and source records attributed to R Lahti.

18 recordsLinked to original sources
PubMed1992

Leukocytes as immunosensors: an immunoassay without labelled reagents.

A novel quantitative nonlabel immunoassay is described. It is based on the recognition of antigen-antibody complexes by the Fc-receptors of phagocytic leukocytes and the subsequent activation of these cells. Activation which is proportional to the amount of immune complexes present can be detected by measuring the intensity of chemiluminescence emitted by the activated cells. In addition to determinations of an antigen and an antibody, the binding capacity of complement to antigen-antibody complexes can be estimated.

Antigen-Antibody Complex↗
PubMed1992

Evolutionary conservation of the active site of soluble inorganic pyrophosphatase.

Soluble inorganic pyrophosphatases (PPases) are essential enzymes that are important for controlling the cellular levels of inorganic pyrophosphate (PPi). Although prokaryotic and eukaryotic PPases differ substantially in amino acid sequence, recent evidence now demonstrates clearly that PPases throughout evolution show a remarkable level of conservation of both an extended active site structure, which has the character of a mini-mineral, and a catalytic mechanism. PPases require several (three or four) Mg2+ ions at the active site for activity and many of the 15-17 fully conserved active site residues are directly involved in the binding of metal ions. Each of the eight microscopic rate constants that has been evaluated for the PPases from both Escherichia coli and Saccharomyces cerevisiae is quite similar in magnitude for the two enzymes, supporting the notion of a conserved mechanism.

Amino Acid Sequence↗
PubMed1991

Transcriptional activation of yeast nucleotide biosynthetic gene ADE4 by GCN4.

The yeast transcriptional regulator protein GCN4 harbors the bZIP DNA binding motif, which is common to a family of DNA-binding proteins in eukaryotic organisms from yeast to man. GCN4 and the mammalian activator protein AP-1 (jun/fos) regulate transcription by binding the same consensus DNA sequence ATGA (C/G)TCAT. GCN4 positively regulates the production of precursors of protein synthesis in yeast cells in response to the environmental signal "amino acid starvation." We find three GCN4 responsive elements (GCREs) in the 5'-flanking region of the purine biosynthetic gene ADE4 and demonstrate that GCN4 efficiently activates transcription of ADE4. Two GCREs are essential to synergistically activate ADE4 transcription by binding GCN4. The distal GCRE1 is also required for basal transcription of ADE4. Therefore, transcription factor GCN4 affects, in addition to protein biosynthesis, also nucleotide biosynthesis and, comparable to its mammalian counterpart AP-1, has a more general function within the yeast cell than previously assumed.

Base Sequence↗
PubMed1991

Genetic engineering of Escherichia coli inorganic pyrophosphatase. Tyr55 and Tyr141 are important for the structural integrity.

Two tyrosines are supposed to be essential for the activity and to participate in the stabilization of Escherichia coli inorganic pyrophosphatase (PPiase) against heat denaturation [Samejima, T., Tamagawa, Y., Kondo, Y., Hachimori, A., Kaji, H., Takeda, A. and Shiroya, Y. (1988) J. Biochem. (Tokyo) 103, 766-772]. To locate these two tyrosines in the amino acid sequence, we substituted all the eight tyrosines of E. coli PPiase with phenylalanine and studied the properties of these YF mutant PPiases. Interestingly, substitution of the tyrosines (Tyr51, Tyr55 and Tyr141) conserved with the amino acid sequence of yeast PPiase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K. and Cooperman, B. (1990) Biochim. Biophys. Acta 1038, 338-345] exerted the most drastic effects on the structure and activity of E. coli PPiase. PPiase variants YF51, YF55 and YF141 had 64%, 7% and 22% of the wild-type PPiase activity, respectively. Furthermore, PPiase variant YF141 had an increased sensitivity to heat denaturation, whereas mutant PPiase YF55 displayed a profound conformational change, as demonstrated by the binding of the fluorescent dye 9-(diethylamino)-5H-benzo(alpha) phenoxazine-5-one (Nile red) that monitors the hydrophobicity of protein surfaces. None of the tyrosines of E. coli PPiase seem to be essential for catalysis, but Tyr55 and Tyr141 are important for the structural integrity of E. coli PPiase.

Amino Acid Sequence↗
PubMed1991

Characterization of the 5' flanking region of the Escherichia coli ppa gene encoding inorganic pyrophosphatase: mutations in the ribosome-binding site decrease the level of ppa mRNA.

We have previously cloned and sequenced the ppa gene, encoding inorganic pyrophosphatase (PPase), of Escherichia coli K12 [Lahti, R., Pitkäranta, T., Valve, E., Ilta, I., Kukko-Kalske, E. & Heinonen, J. (1988) Journal of Bacteriology 170, 5901-5907]. In this work mutations were constructed in the 5' flanking region of E. coli ppa and the effect on expression was determined. The minimum length of the fully active ppa5' flanking region was shown to be 117 bp. Further deletion decreased the activity, and upon deletion to nucleotide -37 the promoter activity was totally lost. A clear point of inflection was observed in the inactivation upon deletion over the nucleotide -50. This is consistent with the fact that by binding to promoters RNA polymerase holoenzyme generally covers the -50 to +20 region in E. coli genes. When the -35 sequence of ppa, AAGACA, was mutated to AAAACA, ppa expression, as measured by PPase production, decreased to 20% of the wild-type, whereas by the change of the -10 sequence, TATAAT, to TTTAAT or TATAAA, the ppa gene was totally inactivated. Furthermore, when the ribosome-binding site (RBS) sequence, AGGAAA, was altered to AAGAAA, PPase production decreased to 19% of the wild-type. Surprisingly, when the RBS sequence was mutated to the consensus RBS sequence, AGGAGG, the intracellular levels of both ppa mRNA and PPase decreased drastically. The implications of these results are discussed.

Base Sequence↗
PubMed1991

Interaction of pyrophosphate ion with di-, tri- and tetra-amines in aqueous solution: a potentiometric and calorimetric study.

Successive macroscopic pKa values have been determined potentiometrically for the conjugate acids of spermidine (4-azaoctane-1,8-diamine), spermine (4,9-diazadodecane-1,12-diamine) and some acylic diamines at various ionic strengths in the absence and presence of tetrasodium pyrophosphate. The stability of the amine/pyrophosphate adducts have been estimated on the basis of effects of the pyrophosphate ion on the apparent acidity constants of the amines. Stoichiometry and thermodynamics of adduct formation have been elucidated from microcalorimetric measurements.

Amines↗
PubMed1990

A site-directed mutagenesis study on Escherichia coli inorganic pyrophosphatase. Glutamic acid-98 and lysine-104 are important for structural integrity, whereas aspartic acids-97 and -102 are essential for catalytic activity.

Analysis of the conservation of functional residues between yeast and Escherichia coli inorganic pyrophosphatases (PPases) suggested that Asp-97, Glu-98, Asp-102, and Lys-104 are important for the action of E. coli PPase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K., & Cooperman, B. S. (1990) Biochim. Biophys. Acta 1038, 338-345]. We replaced these four residues by oligonucleotide-directed mutagenesis, giving variant PPases DV97, DE97, EV98, DV102, DE102, KI104, and KR104. PPase variants DV97, DV102, and KI104 had no enzyme activity, whereas PPase variants DE97, EV98, DE102, and KR104 had 22%, 33%, 3%, and 3% of the wild-type PPase activity, respectively. This suggests that Asp-97, Asp-102, and Lys-104 are essential for the catalytic activity of E. coli PPase. PPase variants DV98 and KR104 also had an increased sensitivity to heat denaturation; incubation of these mutant PPases at 75 degrees C for 15 min in the presence of 5 mM magnesium ion decreased the activity to 20% and 1%, respectively, of the initial value while 74% of the activity was observed with wild-type PPase. Furthermore, these thermolabile mutant PPases displayed the most profound conformational changes of the PPase variants examined, as demonstrated by the binding of the fluorescent dye Nile red that monitors the hydrophobicity of protein surfaces. Accordingly, Glu-98 and Lys-104 seem to be important for the structural integrity of E. coli PPase.

Amino Acid Sequence↗
PubMed1979

Aeration sensitizes Streptococcus faecalis to hydroxyurea.

Hydroxyurea in up to 60 mM concentration did not inhibit growth or DNA synthesis in nonaerated cultures of Streptococcus faecalis ATCC 8043. In contrast, in cultures aerated by shaking already 1 mM hydroxyurea decreased the rate of net DNA synthesis and in higher concentrations of the drug the growth of the total cell mass also slowed down and the number of cells per chain increased from 1-2 to 10. The differential rate of DNA synthesis, but not the growth of the total cell mass, could be restored almost to the control level by adding thymidine to the medium. Thus there are at least two targets for hydroxyurea in the cells of S. faecalis grown in aerated cultures.

Air↗
PubMed1979

Post-mortem inflation, radiography, and fixation of human lungs. A method for radiological and pathological correlations and morphometric studies.

A method is described for correlative radiological and pathological studies of the lung post mortem, including radiography after air inflation, fixation with formalin-polyethylene glycol-alcohol solution, air drying and systematic histological sampling of lung tissue. For morphometric studies the total lung capacity during life is determined from radiograms and compared with the volume of the excised lung after fixation. The method is simple, relatively inexpensive, and with some experience gives good results both in radiography and histology. With this method the test lungs seemed to assume during fixation a fairly constant volume, a little higher than functional residual capacity.

Aged↗
PubMed1979

Comparison of ultrasound and roentgen examination of biliary tract.

The relative merits of gray-scale ultrasound and roentgen examinations of biliary tract diseases have been compared. Sonography was able to give a definite diagnosis in 74 of 95 roentgenologically unclear cases, and roentgen examination gave a diagnosis in 70 out of 105 cases unclear at sonography.

Biliary Tract Diseases↗
PubMed1978

Lipids in the pulmonary circulation after fatal trauma. A postmortem study.

Lipids in the pulmonary circulation were determined biochemically and histologically in seven patients (group A) who died following multiple blunt trauma and in five patients (group B) who died of some other traumatic or nontraumatic cause. Autopsy was performed on average 4.5 hours after death. Blood samples were collected from the right ventricle (RV) and the left atrium (LA). The vasculature of the left lung was perfused in a retrograde direction. The inferior lobe of the right non-perfused lung was examined histologically. Intravascular pulmonary fat was observed in 4/6 cases in group A and in 1/5 cases in group B. No intravascular fat was observed in the tissue samples of brain, liver or kidney. The concentration of free fatty acids (FFA) in blood from RV was higher than in blood from LA both in group A (P is less than 0.01) and in group B (P is less than 0.05). The considerable difference in concentrations of FFA between RV and LA and the low concentration of FFA in the lung perfusate may be due to some kind of "fixation" of FFA in the lung tissue, possibly connected with the aetiological factors of the fat embolism syndrome (FES).

Adult↗
PubMed1978

Family studies of ocular manifestations in arthritis.

To determine the hereditary and clinical patterns, nine patients from three families with different systemic and ocular rheumatoid diseases were examined ophthalmologically and medically. Three types of HLA-B27 associated anterior uveitis were seen. While HLA-B27 linked genes predispose the carrier to acute anterior uveitis (AAU) frequently recurring or chronic anterior uveitis may develop if an immune-complex disease such as Rheumatoid arthritis coexists. Hereditary factors may dispose patients to rheumatoid episcleritis, scleritis and keratitis.

Adolescent↗
PubMed1977

Acute anterior uveitis and HLA-B27 in families.

To determine the hereditary pattern of acute anterior uveitis (AAU) 12 families with 2 cases of AAU in each family were given a routine eye examination which included serological determination of HLA antigens. Members of four families underwent x-ray examination of the lumbosacral spine. Of the 23 cases with AAU examined, 19 had the antigen B27. The genes determining B27 and a2 were inherited together in 14 instances and the gene determining Cwl in 10 of these 14. The lymphocytes of the HLA identical siblings of the three families studied by the mixed lymphocyte culture test did not react against each other. Sacroilities was seen in 62.5% of cases with familial AAU and in 58.6% of their relatives. It is concluded that HLA-B27 is linked with genes predisposing the carrier to familial AAU, which is closely related to the rheumatic group of diseases.

Acute Disease↗
PubMed1975

Blood lipid changes in musculoskeletal trauma of the rabbit.

After multiple long bone fractures, signs of excessive fat transport were observed in rabbits. Staining of albumen in the lipoprotein electrophoresis suggests an increased free fatty acid mobilisation. During the five day experimental period the beta lipoproteins increased while the alpha1 lipoproteins decreased. On the second post-traumatic day, the proportion of pre- beta lipoproteins increased simultaneously with a four-fold increase in plasma triglycerides. Some increase in cholesterol was also observed. An augmented de novo synthesis of fat is proposed as an explanation for part of the increased post-traumatic lipid transport. The changes were compared with a simultaneous occurrence of fat in the lung and kidney.

Animals↗