Glutaraldehyde erythropoietin protects kidney in ischaemia/reperfusion injury without increasing red blood cell production.

BACKGROUND AND PURPOSE: Recombinant human erythropoietin (rHuEPO) is currently the mainstay of renal anaemia treatment. Recently, rHuEPO has been shown to provide pleiotrophic tissue protection in various pathological conditions. However, the benefits of rHuEPO beyond anaemia treatment are limited because it increases red blood cell mass. Carbamylated erythropoietin (CEPO) is the first rHuEPO derivative that lacks erythropoietic activity but retains tissue protection properties. Since carbamylation targets lysine residues on rHuEPo, we hypothesized that targeted lysine modifications of rHuEPO may result in a novel non-erythropoietic erythropoietin. EXPERIMENTAL APPROACH: rHuEPO was subjected to various targeted lysine modifications. In vitro cytoprotection and apoptosis were evaluated using P19 and HEK293 cells. In vivo erythropoiesis was performed by administering the derivatives to animals for 2 weeks. Renoprotection was tested on an ischaemia/reperfusion (I/R) model. KEY RESULTS: We synthesized a novel derivative, a glutaraldehyde erythropoietin (GEPO). This construct abolished in vivo erythropoiesis. Biochemical characterization showed that GEPO was more electrostatically negative than rHuEPO. Immunoprecipitation experiments revealed that GEPO bound to the IL3RB/EPOR heterotrimeric receptor and ameliorated cellular apoptosis via the activation of Bcl-2. Notably, Bcl-2 activation was suppressed by the JAK2 inhibitor, tyrphostin AG490. In vivo experiments showed that GEPO also ameliorated kidney damage due to I/R injury both functionally and histologically. CONCLUSIONS AND IMPLICATIONS: Herein, we describe a novel lysine-modified rHuEPO, glutaradehyde-EPO (GEPO), obtained from a simple reaction. This derivative has no erythropoietic properties but retains cell-protective characteristics both in vitro and in vivo, with promise for future use as an adjunctive treatment of kidney disease.

Pulmonary MR angiography with contrast agent at 4 Tesla: a preliminary result.

In this study, pulmonary MR angiography (MRA) using a tailored coil at 4 Tesla in conjunction with an intravenous injection of contrast agent is described. Three-dimensional gradient-echo images were obtained during the intravenous injection of 0.05, 0.1, and 0.2 mmol/kg body weight of gadodiamide to investigate the signal enhancement effect of the contrast agent in pulmonary arteries qualitatively and quantitatively. In the qualitative analysis, the subsegmental branches were visualized on every dose. In the quantitative analysis, the average contrast-to-noise ratios (CNRs) of the main pulmonary arteries increased in a dose-dependent manner. However, the CNRs of segmental arteries did not increase as the dose of contrast agent increased, as observed at 1.5 Tesla MRI. These observations demonstrate the feasibility of delineating the pulmonary vasculature using a contrast agent; however, our results also suggest possible high-field-related disabilities that need to be overcome before high-field (> or =4 Tesla) MRI can be used to full advantage.

Hyperpolarized noble gas MR imaging of the lung: potential clinical applications.

Hyperpolarized noble gases are a new class of MR contrast agent. Since the first hyperpolarized gas MR images of the lung were reported, there has been considerable interest in using hyperpolarized gas to obtain high spatial and temporal resolution images of the air spaces of the lung. In addition to static images of lung ventilation, new techniques are being developed using hyperpolarized gas to obtain dynamic, diffusion and oxygen concentration images of the lung. In this article, we review the potential clinical applications of pulmonary hyperpolarized gas MRI and discuss the preliminary findings in a variety of lung diseases. Hyperpolarized gas MRI has the potential to provide a comprehensive morphologic and functional assessment of the lung.

Structural basis for pulmonary functional imaging.

An understanding of fine normal lung morphology is important for effective pulmonary functional imaging. The lung specimens must be inflated. These include (a) unfixed, inflated lung specimen, (b) formaldehyde fixed lung specimen, (c) fixed, inflated dry lung specimen, and (d) histology specimen. Photography, magnified view, radiograph, computed tomography, and histology of these specimens are demonstrated. From a standpoint of diagnostic imaging, the main normal lung structures consist of airways (bronchi and bronchioles), alveoli, pulmonary vessels, secondary pulmonary lobules, and subpleural pulmonary lymphatic channels. This review summarizes fine radiologic normal lung morphology as an aid to effective pulmonary functional imaging.

Comparison of short inversion time inversion recovery (STIR) and fat-saturated (chemsat) techniques for background fat intensity suppression in cervical and thoracic MR imaging.

The purpose of this study was to compare short inversion time inversion recovery (STIR) fast spin-echo (FSE), and fat-saturated T2-weighted FSE sequences in terms of uniformity of fat suppression and lesion conspicuity for magnetic resonance (MR) imaging of the neck and thorax. STIR FSE and fat-saturated T2-weighted FSE images were scored for uniformity of fat suppression (n = 40) and lesion conspicuity (n = 35). Five-point rank score analyses were utilized by three experienced radiologists. The mean scores of STIR and fat-saturated FSE techniques for uniformity of fat suppression were 4.3 and 2.3, respectively (P < 0.0001). The mean scores of STIR and fat-saturated FSE techniques for lesion conspicuity were 4.2 and 3.5, respectively (P < 0.0001). Insufficient fat suppression was prominent in the mandible, supraclavicular region, anterior mediastinum, epipericardial fat, and subdiaphragmatic fat. In addition, fat-saturated T2-weighted FSE showed inadvertent water suppression in 25%. The STIR FSE technique was superior to the fat-saturated FSE technique for cervical and thoracic MR imaging.

Lbx1 is required for muscle precursor migration along a lateral pathway into the limb.

In mammalian embryos, myogenic precursor cells emigrate from the ventral lip of the dermomyotome and colonize the limbs, tongue and diaphragm where they differentiate and form skeletal muscle. Previous studies have shown that Pax3, together with the c-Met receptor tyrosine kinase and its ligand Scatter Factor (SF) are necessary for the migration of hypaxial muscle precursors in mice. Lbx1 and Pax3 are co-expressed in all migrating hypaxial muscle precursors, raising the possibility that Lbx1 regulates their migration. To examine the function of Lbx1 in muscle development, we inactivated the Lbx1 gene by homologous recombination. Mice lacking Lbx1 exhibit an extensive loss of limb muscles, although some forelimb and hindlimb muscles are still present. The pattern of muscle loss suggests that Lbx1 is not required for the specification of particular limb muscles, and the muscle defects that occur in Lbx1(-/-) mice can be solely attributed to changes in muscle precursor migration. c-Met is expressed in Lbx1 mutant mice and limb muscle precursors delaminate from the ventral dermomyotome but fail to migrate laterally into the limb. Muscle precursors still migrate ventrally and give rise to tongue, diaphragm and some limb muscles, demonstrating Lbx1 is necessary for the lateral, but not ventral, migration of hypaxial muscle precursors. These results suggest that Lbx1 regulates responsiveness to a lateral migration signal which emanates from the developing limb.

Separation of human cerebrospinal fluid proteins by capillary isoelectric focusing in the absence of denaturing agents.

Conditions of capillary isoelectric focusing (CIEF) to separate human cerebrospinal fluid (CSF) proteins were examined referring to those which we have established for the separation of human plasma/serum proteins. Since the average protein concentration in CSF is about 1/200 of plasma and the salt concentration is at almost the same level as plasma, desalting of CSF samples with minimum dilution was a prerequisite for CIEF analysis of CSF proteins. We constructed an apparatus to dialyze CSF at the level of 20-30 microL, this volume being sufficient for 3-4 repeated analyses of the CSF sample. To trace the process of dialysis, a simple device to measure the conductivity of the dialyzate was also constructed. Most of the CIEF conditions for plasma protein analysis could be applied for CSF protein analysis. However, the addition of N,N,N',N'-tetramethylethylenediamine (TEMED) at a suitable concentration was necessary to improve the resolution of basic proteins (IgG region), since some CSF patterns showed peaks of basic proteins which are not obvious in the serum of the same patient. About 70 peaks and shoulders of CSF proteins could be detected by the established CIEF technique. The results of CIEF analysis of CSF samples suggested that the technique will be useful as a survey method to detect specific proteins in CSF, which might relate to disorders in the central nervous system.

[Ultrasonogram of carotid artery in endoarterectomy patients].

Ultrasonograms of the carotid artery were compared with the intraoperative finding of atheroma plaque in terms of stenosis, fragility, ulceration and calcification. Soft type, intermediate type and these mixed type plaques were fragile plaques in almost all cases. All hard type plaques were tough plaques. The detection of stenosis and calcification was satisfactory, however that of ulceration was not sufficient. The stenotic lesions frequently existed in bilateral carotid arteries and the intima was easy to become thick even after the endoarterectomy. The ultrasonogram was thought to be the essential study for the carotid artery lesions.

A novel activating mutation in calcium-sensing receptor gene associated with a family of autosomal dominant hypocalcemia.

Autosomal dominant hypocalcemia (ADH), caused by activating mutations of the calcium-sensing receptor (CaSR), is characterized by hypocalcemia with an inappropriately low concentration of PTH. Among 11 missense mutations of CaSR reported to date in patients with ADH or sporadic hypocalcemia, functional properties of 8 mutant CaSRs were characterized. Here, we describe a novel mutation of CaSR and its functional property in a family with ADH. The 41-yr-old male proband had asymptomatic hypocalcemia with a history of recurrent nephrolithiasis. His father had asymptomatic hypocalcemia, but his mother was normocalcemic. PCR-single strand conformation polymorphism and sequencing revealed that both the proband and the father had a novel heterozygous mutation in CaSR gene that causes lysine to asparagine substitution at codon 47 (K47N), which is in the extracellular domain of CaSR, like 6 of 11 known activating mutations. Using HEK293 cells transfected with wild-type or K47N CaSR complementary DNA, the intracellular Ca2+ concentration was assessed in response to changes in the extracellular Ca2+ concentration. The EC50 of the mutant CaSR for the extracellular Ca2+ concentration was 2.2 mmol/L and was significantly lower than that of wild-type (3.7 mmol/L). These results confirm that this mutation is responsible for ADH in this family. The fact that several inactivating mutations in familial hypocalciuric hypercalcemia occur in amino acid around K47 suggests the importance of the N-terminal portion of the receptor in extracellular Ca sensing.

CBFB/MYH11 fusion transcripts in a case of acute myelogenous leukemia (M1) with partial deletion of the long arm of chromosome 16.

Pericentric inversion of chromosome 16 [inv(16)(p13q22)] is seen in patients with acute myelomonocytic leukemia with bone marrow eosinophilia. This inversion juxtaposes the MYH11 gene on p13 and the CBFB gene on q22, resulting in the formation of a chimeric mRNA transcript. We describe a patient with acute myelogenous leukemia (M1), with del(16)(q22), who expressed the chimeric transcript. Reverse transcriptase polymerase chain reaction and the sequencing of its product showed fusion of 5'CBFB at position 495 to 3'MYH11 at position 1201. To our knowledge, this is the first report of an AML (M1) case with del(16) and CBFB/MYH11 rearrangement.

Secretion of active human lysozyme by Acremonium chrysogenum using a Fusarium alkaline protease promoter system.

We constructed expression vectors for Acremonium chrysogenum using a Fusarium alkaline protease promoter region and tested their potential as secretion systems for foreign proteins using the human (h)-lysozyme gene as an indicator. The gene encoding h-lysozyme was linked to the coding region of (1) the carboxy terminal of the alkaline protease pre peptide, (2) the carboxy terminal of the prepro peptide, (3) three amino acids of the mature protein preceded by the prepro peptide and (4) the carboxy terminal of chicken lysozyme signal peptide, inserted into the genomic DNAs of A. chrysogenum and expressed under the control of the alkaline protease promoter. The transformants of A. chrysogenum with each of these plasmids secreted enzymatically active h-lysozyme. A maximum yield in excess of 40 mg l-1 was obtained when h-lysozyme was linked to the carboxy terminal of alkaline protease prepro peptide. The majority of the amino terminal sequence of the purified h-lysozyme from the culture supernatant was identical with that of authentic h-lysozyme, but it showed some heterogeneity.

High level expression of Fusarium alkaline protease gene in Acremonium chrysogenum.

We transformed Acremonium chrysogenum with the genomic DNA of the alkaline protease (Alp) from Fusarium sp. S-19-5 including its promoter. Most of the transformants thus obtained produced a large amount of Alp. PCR and Southern hybridization analysis of genomic DNAs from these transformants showed chromosomal integration of the full-length Alp gene. SDS-PAGE analysis of the supernatant from the transformants showed the presence of Fusarium Alp. The amino terminus of the Alp produced in A. chrysogenum was identical to that of native Fusarium Alp. These results indicate that the Alp promoter, signal sequence, and introns functioned correctly in A. chrysogenum. One of the transformants produced more than 4 g of the Alp per liter in a jar fermentor.

A comparison of methods in measurement of effective renal plasma flow and glomerular filtration rate in clinical practice.

Using a single injection clearance method as a reference test, 24-hour creatinine clearance and gamma camera estimation of ERPF and GFR were compared in a sample of the Japanese adult patient population. The reference tests included ERPF with I-131 OIH and GFR with In-111 DTPA, single blood sample, and 1-hour external counting over the heart for each tracer. The 24-hour creatinine clearance and gamma camera methods were equally accurate in estimating GFR or ERPF compared with the clearance methods. The gamma camera methods were less accurate for GFR than ERPF. Among many gamma camera estimation formulas for GFR, the renal Tc-99m DTPA uptake best correlated with the reference GFR (compared to Schlegel's GFR or Gates' GFR). The authors found that the simpler the procedure and data processing, the better the results. Origin of errors in estimating ERPF and GFR, the procedures of choice, patient population studied, quality control achievable in clinical practice, and the clinical situations the clinicians are likely to encounter are discussed.

Angiographic retrieval of foreign bodies in pulmonary artery: a report of three cases.

Three cases of percutaneous retrieval of foreign bodies that migrated into the pulmonary artery are described in this paper. All of the intravascular foreign bodies were attributed to SVC catheters for intravenous hyperalimentation (IVH) therapy, an accidentally cut strip of indwelling SVC catheter in two patients and a fragment of guide wire used for the insertion of the SVC catheter in one patient. They were all successfully removed by fluoroscopic angiographic procedures using a snare loop retriever and grasping forceps. The technical aspects of the procedures are discussed here.

Assessment of drug disposition in the perfused rat brain by statistical moment analysis.

Drug disposition in the brain was investigated by statistical moment analysis using an improved in situ brain perfusion technique. The right cerebral hemisphere of the rat was perfused in situ. The drug and inulin were injected into the right internal carotid artery as a rapid bolus and the venous outflow curve at the posterior facial vein was obtained. The infusion rate was adjusted to minimize the flow of perfusion fluid into the left hemisphere. The obtained disposition parameters were characteristics and considered to reflect the physicochemical properties of each drug. Antipyrine showed a small degree of initial uptake. Therefore, its apparent distribution volume (Vi) and apparent intrinsic clearance (CLint,i) were small. Diazepam showed large degrees of both influx and efflux and, thus, a large Vi. Water showed parameters intermediate between those of antipyrine and those of diazepam. Imipramine, desipramine, and propranolol showed a large CLint,i compared with those of the other drugs. The extraction ratio of propranolol significantly decreased with increasing concentrations of unlabeled propranolol in the perfusion fluid. These findings may be explained partly by the tissue binding of these drugs. In conclusion, the present method is useful for studying drug disposition in the brain.

[Detection of resection line in early gastric cancer].

In patients with multiple gastric cancer and superficial spreading type gastric carcinoma (abbreviated as S.S.C.) accompanied by IIb, the remnant stomach is sometimes cancer positive. Our study population consisted of 44 patients with multiple early gastric cancer and 63 patients with depressed type S.S.C. The gastric mucosa was classified into 3 types according to the surrounding mucosal atrophy, 1) pyloric gland zone, 2) intermediate zone, 3) fundic gland zone. All lesions of multiple early gastric cancer and S.S.C. were located in the pyloric gland zone or the intermediate zone. The depressed type S.S.C. did not invade the fundic gland zone. Therefore, knowledge about the extend++ of surrounding mucosal atrophy is required for the proper diagnosis of early gastric cancer and the surgeon must recognize the relationship between the glandular border line and resection line.