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Biomedical subjects

J Tenhunen

Publications and source records attributed to J Tenhunen.

At least 19 recordsLinked to original sources
PubMed2005

Physiological and morphological responses to water stress in two Acacia species from contrasting habitats.

Container-grown seedlings of Acacia tortilis Forsk. Hayne and A. xanthophloea Benth. were watered either every other day (well watered) or every 7 days (water-stressed) for 1 year in a greenhouse. Total plant dry mass (T(dm)), carbon allocation and water relations were measured monthly. Differences in leaf area (LA) accounted for differences in T(dm) between the species, and between well-watered and water-stressed plants. Reduction in LA as a result of water stress was attributed to reduced leaf initiation, leaf growth rate and leaf size. When subjected to prolonged water stress, Acacia xanthophloea wilted more rapidly than A. tortilis and, unlike A. tortilis, lost both leaves and branches. These differences between species were attributed to differences in the allocation of carbon between leaves and roots and in the ability to adjust osmotically. Rapid recovery in A. xanthophloea following the prolonged water-stress treatment was attributed to high cell wall elasticity. Previous exposure to water stress contributed to water-stress resistance and improved recovery after stress.

Acacia↗
PubMed2003

Analysis of depth profiling data obtained by confocal Raman microspectroscopy.

The nominal depth resolution achieved in confocal Raman microscopy is on the order of a few micrometers. Often, however, the depth resolution is decreased by light refraction at the sample surface. The problem can be avoided with the use of an immersion objective and index matching oils. Through this intervention the instrument point-spread function (PSF) can be assumed to be independent of the depth of focus in the sample, and spatially invariant depth profiles can be acquired. In this work the instrument PSF was determined by measuring a depth profile of a thick uniform sample and calculating the first derivative of the depth profile curve. The first-derivative method was also used to determine sample thickness. Convolution with the PSF makes it possible to simulate the behavior of the instrument with different sample functions. It is also possible to use the instrument PSF to deconvolve depth-profiling data. Deconvolution reduces the blurring effect of the instrument and increases the depth resolution. Deconvolution can also be used in analysis of the sample surface position and in layer structure analysis. In this paper we show how the convolution integral can be used with the immersion sampling technique to determine the PSF and how the sample thickness can be determined.

Algorithms↗
PubMed2001

Characterization of wet massing behavior of silicified microcrystalline cellulose and alpha-lactose monohydrate using near-infrared spectroscopy.

The purpose of this study was to investigate the energetic state of water in silicified microcrystalline cellulose (SMCC) and alpha-lactose monohydrate wet masses using near-infrared (NIR) spectroscopy. The applicability of NIR spectroscopy to studying pharmaceutical wet masses at a wide moisture range was evaluated in comparison with mixer torque rheometry (MTR). With increasing moisture content changes in the physical properties of the samples resulted in an apparent increase in log (1/R) throughout the whole spectrum. The upward displacement of baseline and the relative height of water bands were greatest with materials that had a poor liquid-retention capacity. In the case of SMCC and 1:1 mixture of SMCC and alpha-lactose monohydrate, the height of the baseline-corrected water bands increased linearly at low moisture contents, thereafter achieving a plateau stage. According to the MTR results, the plateau stage of the band heights indicated a capillary state of liquid saturation. The second derivative spectrum was capable of distinguishing monohydrate, absorbed, and adsorbed water, which overlapped in the absorbance spectrum. When water was absorbed to the internal structure of the material (SMCC), the water bands were first seen at higher wavelengths, then followed by a shift to lower wavelengths. When water was only adsorbed onto the surface of the particles (glass ballotini), the water bands were seen directly in the region of bulk water.

Crystallization↗
PubMed2000

Next generation fluidized bed granulator automation.

A system for fluidized bed granulator automation with in-line multichannel near infrared (NIR) moisture measurement and a unique air flow rate measurement design was assembled, and the information gained was investigated. The multivariate process data collected was analyzed using principal component analysis (PCA). The test materials (theophylline and microcrystalline cellulose) were granulated and the calibration behavior of the multichannel NIR set-up was evaluated against full Fourier Transform (FT) NIR spectra. Accurate and reliable process air flow rate measurement proved critical in controlling the granulation process. The process data describing the state of the process was projected in two dimensions, and the information from various trend charts was outlined simultaneously. The absorbence of test material at correction wavelengths (NIR region) and the nature of material-water interactions affected the detected in-line NIR water signal. This resulted in different calibration models for the test materials. Development of process analytical methods together with new data visualization algorithms creates new tools for in-process control of the fluidized bed granulation.

Calibration↗
PubMed2000

In-line moisture measurement during granulation with a four-wavelength near infrared sensor: an evaluation of particle size and binder effects.

Factors affecting in-line near infrared (NIR) moisture measurement with a four-wavelength sensor were evaluated (choice of binder used in granulation liquid and the increase in particle size). An entire NIR spectrum is not necessary for the measurement of water, and often the use of only a few NIR wavelengths around the water band enables reliable and high-speed detection of moisture. Glass ballotini and microcrystalline cellulose (MCC) were used as model test materials. The binders studied were poly[1-(2-oxo-1-pyrrolidinyl)ethylene] (PVP) and gelatin. Full off-line NIR spectra of test materials at different levels of binder solutions were measured. The major spectral features for both the binders were bands around 1700 nm (first overtones CH related stretches) and 2200 nm (combination bands). Gelatin also had an NH band around 1500 nm (first overtones of NH stretches) and combination bands at about 2050 nm. Particle size effects were observed as an increase in spectra baseline. All these factors should be considered when choosing NIR wavelengths used for detection of water with a fixed wavelength set-up. A robust calibration model enables the development of in-process control of wet granulation processes.

Calibration↗
PubMed2000

Effects of dobutamine on splanchnic tissue perfusion during partial superior mesenteric artery occlusion.

OBJECTIVE: To evaluate the effects of dobutamine and fluid treatment on splanchnic hemodynamics and tissue oxygenation during partial superior mesenteric artery occlusion. DESIGN: Prospective, open randomized, full-factorial design. SETTING: University research laboratory. SUBJECTS: Forty-eight female pigs. INTERVENTIONS: In 24 anesthetized pigs (ischemic group), superior mesenteric artery (SMA) blood flow was reduced to 30% from the baseline for 120 mins; 24 pigs (sham group) served as nonischemic controls. The animals were further assigned into four treatment arms. In the control arm, the animals were administered only basic fluid therapy. In the fluid therapy arm, pulmonary artery occlusion pressure was maintained at 10 mm Hg with fluids. In the dobutamine treatment arm, dobutamine hydrochloride was infused at a dose of 10 microg/min/kg. In the combined dobutamine-fluid therapy arm, dobutamine at 10 microg/min/kg was administered and pulmonary artery occlusion pressure was maintained at 10 mm Hg with fluids. MEASUREMENTS AND MAIN RESULTS: Systemic and regional hemodynamics and oxygen transport, as well as jejunal intramucosal pH, intramucosal-arterial PCO2 gradient, and portal venous-arterial lactate gradient were measured. Ischemia did not modify the effects of fluids or dobutamine on systemic hemodynamics and oxygen transport. Dobutamine-treated animals had a higher cardiac index compared with control animals (218 +/- 22 vs. 135 +/- 13 mL/min/kg; p = .012), and the effect was enhanced when dobutamine was combined with fluid treatment (365 +/- 23 mL/ min/kg; p = .019). Fluid treatment alone did not influence cardiac index, whereas it increased SMA blood flow compared with control groups (15 +/- 2 vs. 12 +/- 2 mL/min/kg; p = .023). Dobutamine also decreased the proportion of SMA blood flow of cardiac output compared with control groups (6 +/- 1 vs. 9% +/- 1%; p = .024). Other treatments had no effect on SMA blood flow. Ischemia increased intramucosal-arterial Pco2 gradient to 54.8 +/- 10.7 torr (7.31 +/- 1.43 kPa) (p = .002 vs. sham control) and decreased intramucosal pH to 7.13 +/- 0.06 (p = .028 vs. sham control). In the ischemic animals, dobutamine without fluid therapy reduced intramucosal pH further to 7.00 +/- 0.09 (p = .023 vs. ischemic control) and increased portal venous-arterial lactate gradient (p = .033). CONCLUSIONS: Dobutamine alone worsened splanchnic tissue perfusion during partial superior mesenteric artery occlusion. As compared with fluid treatment alone, the combination of fluid and dobutamine therapy did not improve tissue perfusion.

Animals↗
PubMed1999

Soluble and membrane-bound catechol-O-methyltransferase in normal and malignant mammary gland.

The levels of 26 kDa-soluble (S) and 30 kDa-membrane-bound (MB) catechol-O-methyltransferase (COMT) polypeptides were determined in paired samples from normal and neoplastic breast tissue of 32 patients with breast cancer. Immunohistochemical staining showed that the COMT reaction in normal mammary tissue was restricted to the epithelial cells in the ducti and lobuli, whereas in the tumors a strong reaction was also seen in the malignant cells. The amounts of COMT proteins in tumors could not be correlated with various clinical or pathological parameters. Quantitative immunoblotting analysis revealed that the total amount of COMT proteins in tumors was more than 50% higher than in respective normal samples in 26 out of 32 patients. Five cases showed less than a 50% difference and in one case less COMT was detected in the tumor. In most cases the amount of both S- and MB-COMT forms was increased. The average amount of total COMT was 178 +/- 57 pg/microg total protein in normal tissue and 566 +/- 94 pg/microg total protein in tumor. Respective values for S-COMT were 137 +/- 52 pg/microg total protein in normal tissue and 369 +/- 62 pg/microg total protein in tumor and for MB-COMT 41 +/- 10 and 197 +/- 41 pg/microg total protein, respectively. Analysis of COMT-specific transcripts suggested that the COMT enzyme level in tumors is determined in some cases by transcriptional and in some cases by post-transcriptional mechanisms.

Adult↗
PubMed1998

Angiotensin converting enzyme inhibition has no effect on blood pressure and splanchnic perfusion after cardiac surgery.

PURPOSE: The purpose of this study was to study the effect of the angiotensin-converting enzyme inhibitor, enalaprilat, on blood pressure and splanchnic perfusion after cardiac surgery. MATERIALS AND METHODS: Sixteen patients were studied after coronary artery bypass grafting. After admission to the intensive care unit, a 30-minute baseline measurement of systemic hemodynamics, oxygen transport, and gastric tonometry was performed. In 6 of 10 patients receiving enalaprilat and in each of 6 control patients, regional (splanchnic and leg) blood flows were measured also. After the baseline measurement period, 10 patients received a 0.5 mg bolus of enalaprilat and thereafter an incremental infusion of enalaprilat up to a total dose of 10 mg (mean 8.3; range 4 to 10 mg) was continued to reduce the mean arterial pressure (MAP) to 70 to 80 mm Hg. A 30-minute measurement period was repeated 2 to 3 hours after the first measurement period. In the control group, the second measurement was performed at corresponding time points. RESULTS: Though MAP decreased in the enalaprilat group (enalaprilat 99 +/- 14 mm Hg v 89 +/- 21 mm Hg, P < .05; control 95 +/- 13 mm Hg v82 +/- 10 mm Hg, P = NS) in only 4 of 10 patients was the targeted MAP reduction achieved. No significant changes were observed either in systemic or regional blood flows. Systemic, pulmonary, and femoral vascular resistance indices decreased significantly in both groups. Gastric-arterial PCO2 difference did not change in either groups. Angiotensin-converting enzyme activity decreased in the enalaprilat group (10.0 +/- 2.3 v 1.3 +/- 0.3 U x l(-1), P < .01), but plasma renin and endothelin-1 concentrations did not change in either group. CONCLUSIONS: The effect of enalaprilat on blood pressure was poor and it had no beneficial effects on splanchnic circulation. Renin-angiotensin activation is not a major factor in hypertension and splanchnic perfusion after cardiac surgery.

Aged↗
PubMed1998

Molecular genetic study of Finns with hypoalphalipoproteinemia and hyperalphalipoproteinemia: a novel Gly230 Arg mutation (LCAT[Fin]) of lecithin:cholesterol acyltransferase (LCAT) accounts for 5% of cases with very low serum HDL cholesterol levels.

In an attempt to identify genetic factors underlying extreme alterations of serum HDL cholesterol (HDL-C) concentrations, we examined two probands with HDL-C levels <0.2 mmol/L and subsequently screened two large cohorts of smoking men, one with very low (0.2 to 0.7 mmol/L, n=156) and the other with elevated (1.9 to 3.6 mmol/L, n=160) HDL-C levels, for the newly detected mutations as well as some other mutations proposed to affect HDL-C levels. One of the probands had corneal opacities, microalbuminuria, hypertriglyceridemia, and reduced LDL apoprotein B concentration; the other had anemia and presented with stomatocytosis in his peripheral blood. The first proband was found to be homozygous for a novel LCAT Gly230Arg (LCAT[Fin]) mutation, and the second was homozygous for an Arg399Cys mutation we described previously. Transient expression of the mutant LCAT(Fin) cDNA in COS cells disclosed markedly diminished LCAT enzyme activity. In the low-HDL-C group of men (n=156), 8 carriers of LCAT(Fin) and 1 carrier of the LCAT Arg399Cys were identified. In addition, the frequency of the lipoprotein lipase (LPL) Asn291Ser mutation was significantly (P<.05) higher in the low-HDL-C group (4.8%) than in the high-HDL-C group (1.6%). In addition, we identified 1 carrier of the intron 14G-->A mutation of cholesterol ester transfer protein (CETP) in the high-HDL-C group and subsequently demonstrated cosegregation of the mutant allele with elevated HDL-C levels in the proband's family. In conclusion, we have identified a novel LCAT gene Gly230Arg mutation (LCAT[Fin]), which, together with the LPL Asn291Ser mutation, represents a relatively common genetic cause of diminishing HDL-C levels, at least among Finns. This article also reports occurrence of a CETP mutation in subjects having non-Japanese roots.

Adult↗
PubMed1997

Expression and regulation of the human and mouse aspartylglucosaminidase gene.

Aspartylglucosaminidase (AGA) is a lysosomal enzyme that catalyzes one of the final steps in the degradation of N-linked glycoproteins. Here we have analyzed the tissue-specific expression and regulation of the human and mouse AGA genes. We isolated and characterized human and mouse AGA 5'-flanking sequences including the promoter regions. Primer extension assay revealed multiple transcription start sites in both genes, characteristic of a housekeeping gene. The cross-species comparison studies pinpointed an approximately 450-base pair (bp) homologous region in the distal promoter. In the functional analysis of human AGA 5' sequence, the critical promoter region was defined, and an additional upstream region of 181 bp exhibiting an inhibitory effect on transcription was identified. Footprinting and gel shift assays indicated protein binding to the core promoter region consisting of two Sp1 binding sites, which were sufficient to produce basal promoter activity in the functional studies. The results also suggested the binding of a previously uncharacterized transcription factor to a 23-bp stretch in the inhibitory region.

Animals↗
PubMed1997

Expression and intracellular localization of catechol O-methyltransferase in transfected mammalian cells.

The intracellular localization of soluble and membrane-bound isoforms of rat and human catechol O-methyltransferase (COMT) was studied by expressing the recombinant COMT proteins either separately or together in mammalian cell lines (HeLa and COS-7 cells) and in rat primary neurons. The distribution of soluble and membrane-bound COMT enzyme was visualized by immunocytochemistry. For comparison, the localization of native COMT was studied in rat C6 glioma cells by immunoelectron microscopy. Staining of cells expressing membrane-bound COMT with a COMT-specific antiserum revealed an immunofluorescence signal in intracellular reticular structures and in the nuclear membrane. Double-staining of the cells with antisera against proteins specific for the rough endoplasmic reticulum indicated that they colocalized with membrane-bound COMT, suggesting that it resided in the endoplasmic reticulum. Notably, no COMT-specific fluorescence of plasma membranes was detected. The signal in the endoplasmic reticulum was also evident in the cells expressing both recombinant COMT forms. Intracellular native COMT reaction was detected by immunoelectron microscopy in rat C6 glioma cells and an intense cytoplasmic signal was seen in the primary neurons infected with the recombinant Semliki Forest virus. The cells expressing recombinant soluble COMT revealed intense nuclear staining together with diffuse cytoplasmic immunoreactivity, suggesting that a part of soluble COMT is transported to nuclei. Western blotting from rat liver and brain revealed soluble COMT in the nuclei. Enzyme activity measurements from liver cytoplasmic and nuclear fractions suggested that about 5% of the soluble COMT resided in nuclei. The intracellular localization of both COMT forms implies that COMT acts in the cytoplasm and possibly also in the nuclear compartment, and that the physiological substrates of COMT enzymes may have to be internalized before their methylation by COMT.

Animals↗
PubMed1996

Characterization of the rat catechol-O-methyltransferase gene proximal promoter: identification of a nuclear protein-DNA interaction that contributes to the tissue-specific regulation.

The methylating enzyme catechol-O-methyltransferase (COMT) is an important inactivator of substrates containing catechol-structure, such as catechol neurotransmitters and hormones. In previous studies, the rat COMT gene has been cloned and characterized, and it has been shown that the two COMT polypeptides, S- and MB-COMT, are expressed from one gene by cooperation of two separate promoters. One promoter, P2, functions constitutively, whereas the other, the proximal P1 promoter, is regulated in a tissue-specific manner. In this report, a more detailed analysis of the rat P1 promoter is presented. By using reporter gene constructs, it is shown that upstream sequences of the P1 promoter contain several regions that modulate the expression either positively or negatively. These experiments also show that the region between the MB- and S-ATG translation initiation codons is indispensable for the activity of this promoter. Analysis of this region by DNase I footprinting and gel retardation assays identified the presence of several DNA elements with SP1 and NF1 recognition site homologies that bound both liver and brain nuclear proteins. However, one 11-nucleotide-long DNA region containing an overlapping consensus binding sequence for CREB and C/EBP-like factors reacted only with the liver nuclear lysate. Supershift experiments suggest that the transcription factor C/EBPalpha mediates the tissue-specific expression of the rat COMT P1 promoter.

Animals↗
PubMed1994

Genomic organization of the human catechol O-methyltransferase gene and its expression from two distinct promoters.

Human genomic DNA fragments containing catechol O-methyltransferase (COMT) sequences were isolated and the exon-intron structure analysed by sequencing, PCR and comparing to the human COMT cDNA sequences. The gene contains six exons, of which exons 1 and 2 are noncoding. MB-ATG and S-ATG codons, responsible for the initiation of translation of the membrane-bound (MB) and soluble (S) forms of the enzyme, are located in exon 3. Two distinct COMT-specific transcripts, 1.3 kb and 1.5 kb, were detected in various human tissues and cell lines. Different quantities of the shorter COMT-specific mRNA in the tissues studied suggest a tissue-specific regulation of the COMT gene at transcriptional level. Mapping of the 5' ends of the COMT mRNAs showed that transcription initiates at multiple sites in two separate DNA regions, which are preceded by functional promoter sequences. The proximal promoter (P1), located between the two translation initiation codons and extending approximately 200 bp upstream of the MB-ATG initiation codon, apparently gives rise to the 1.3-kb S-COMT mRNA (S-mRNA). The distal promoter (P2) is located in a DNA fragment in front of and partly overlapping the transcription-start region of the 1.5-kb transcript, suggesting that it controls the expression of this MB-mRNA. Similarities between the rat and human COMT gene promoters are analyzed.

Animals↗
PubMed1993

Production of rat soluble and membrane-bound catechol O-methyltransferase forms from bifunctional mRNAs.

In the rat, the catechol O-methyltransferase (COMT) gene has been found to contain two promoters, P1 and P2. This organization enables the gene to produce a soluble (S-COMT) and a membrane-associated (MB-COMT) protein by using two in-frame ATG initiation codons (S- and MB-ATG). The P1 promoter expresses a 1.6 kb transcript (S-mRNA) which codes for the S-COMT polypeptide only. Here we demonstrate that the P2 promoter controls the expression of alternatively spliced 1.9 kb transcripts (MB-mRNA) which differ by a 27-nucleotide region immediately upstream of the MB-AUG codon. The presence of the 27-base sequence alters the nucleotide at position -3 from G to C, thereby changing the translation initiation context of the MB-AUG codon. Expression experiments in COS-7 cells using full-length COMT cDNAs showed that this alteration affected the initiation of the translation of the MB-AUG and consequently changed the relative amounts of MB- and S-COMT polypeptides produced. No proteolytic cleavage of the MB-COMT form to S-COMT was detected in in vitro or in vivo pulse-chase experiments. We conclude that the bifunctional 1.9 kb mRNAs are able to produce both S-COMT and MB-COMT polypeptide by the leaky scanning mechanism of translation initiation.

Alternative Splicing↗
PubMed1993

Structure of the rat catechol-O-methyltransferase gene: separate promoters are used to produce mRNAs for soluble and membrane-bound forms of the enzyme.

The enzyme catechol-O-methyltransferase (COMT) catalyzes the inactivation of catechol-containing molecules by methylation. The cDNAs for the rat and human COMT have recently been cloned and recombinant proteins expressed in prokaryotic and eukaryotic cells. We describe here the structure of the rat COMT gene and its 5'-flanking sequences. The gene spans at least 13 kb and is composed of 5 exons, the first one noncoding. The two ATG codons for the initiation of translation of the membrane-bound (MB-COMT) and soluble (S-COMT) forms of the enzyme reside in the second exon. The gene expresses two mRNA species of 1.6 kb and 1.9 kb that have different tissue distributions. The expression of the transcripts is regulated by at least two promoters, P1 and P2. The P1 promoter expresses the shorter transcript in a tissue-specific manner and is located between the ATG codons in the coding region of the longer transcript. The P2 promoter is constitutive and responsible for the expression of the longer transcript. The shorter 1.6-kb mRNA (S-mRNA) produces only the S-COMT polypeptide, whereas the longer 1.9-kb mRNA (MB-mRNA) is able to direct synthesis of both forms of the COMT enzyme.

Animals↗