Open flow microperfusion: pharmacokinetics of human insulin and insulin detemir in the interstitial fluid of subcutaneous adipose tissue.

AIMS: To compare the time profile of insulin detemir and human insulin concentrations in the interstitial fluid (ISF) of subcutaneous adipose tissue during constant i.v. infusion and to investigate the relationship between the pharmacokinetics of both insulin molecules in plasma and the ISF of subcutaneous adipose tissue. METHODS: During a 6-h hyperinsulinaemic-euglycaemic clamp (plasma glucose level 8 mmol/l) human insulin (21 and 42 pmol/min/kg) or insulin detemir (209 and 417 pmol/min/kg) were infused i.v. in eight rats per dose level. Open flow microperfusion (OFM) was used to continuously assess interstitial insulin concentrations in subcutaneous adipose tissue. RESULTS: At the lower infusion rate, insulin detemir appeared significantly later in the ISF than in the plasma (p < 0.05) and also appeared later in the ISF relative to human insulin (p < 0.005). CONCLUSIONS: By using OFM we were able to monitor albumin-bound insulin detemir directly in the ISF of subcutaneous tissue and confirm its delayed transendothelial passage to a peripheral site of action.

Accumulation of SNAP25 in mouse gustatory and somatosensory cortices in response to food and chemical stimulation.

Food intake stimuli, including taste, somatosensory, and tactile stimuli, are received by receptors in the oral cavity, and this information is then transferred to the cerebral cortex. Signals from recently ingested food during the weaning period can affect synaptic transmission, resulting in biochemical changes in the cerebral cortex that modify gustatory and somatosensory nervous system plasticity. In this study, we investigated the expression patterns of molecular markers in mouse gustatory and somatosensory cortices during the weaning period. The expression of synaptosomal-associated protein 25 (SNAP25), a component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, was increased in the insular and somatosensory cortices at postnatal week 3 compared to postnatal week 2. Additionally, SNAP25 protein in the cerebral cortex accumulated in weaning mice fed solid food but not in mice fed only mother's milk at the weaning stage. Chemical stimulation by saccharin or capsaicin at the weaning stage also increased SNAP25 immunoreactivity in the insular or somatosensory cortical area, respectively. These results suggest that recently ingested chemical signals in the oral cavity during weaning increase the accumulation of SNAP25 in the gustatory and somatosensory cortices and promote neural plasticity during the development of the gustatory and somatosensory nervous systems.

Insulin detemir is a fully efficacious, low affinity agonist at the insulin receptor.

AIM: To compare the properties of insulin detemir with human insulin or insulin aspart in various in vitro and in vivo experiments, thereby highlighting the importance of performing dose-response studies when investigating insulin analogues, in this study specifically insulin detemir. METHODS: Displacement of membrane-associated insulin receptors from human and rat hepatocytes, and from Chinese Hamster Ovary cells over-expressing human insulin receptor (CHO-hIR) at varying albumin concentrations is measured. Lipogenesis in primary rat adipocytes over time and the effects in the simultaneous presence of insulin detemir and human insulin or insulin aspart are assessed. The hyperinsulinaemic euglycaemic clamp technique in rats is used to establish dose-response curves for multiple metabolic endpoints and to investigate the effects of the simultaneous presence of insulin detemir and human insulin. RESULTS: Both in vitro and in vivo, insulin detemir shows full efficacy and right-shifted parallel dose-response curves compared with human insulin. The potency estimates are different between the in vivo and in vitro conditions and among different in vitro conditions, that is the potency decreases in vitro with increasing albumin concentration. The effects of insulin detemir and human insulin are additive both in vitro and in vivo. CONCLUSIONS: Insulin detemir is fully efficacious compared with human insulin on all metabolic endpoints measured in vitro and in vivo. The fact that the potency estimates are method-dependent emphasizes the importance of establishing full dose-response relationships when characterizing insulin detemir.

Glucagon receptor antagonism improves islet function in mice with insulin resistance induced by a high-fat diet.

AIMS/HYPOTHESIS: Increased glucagon secretion predicts deterioration of glucose tolerance, and high glucagon levels contribute to hyperglycaemia in type 2 diabetes. Inhibition of glucagon action may therefore be a potential novel target to reduce hyperglycaemia. Here, we investigated whether chronic treatment with a glucagon receptor antagonist (GRA) improves islet dysfunction in female mice on a high-fat diet (HFD). MATERIALS AND METHODS: After 8 weeks of HFD, mice were treated with a small molecule GRA (300 mg/kg, gavage once daily) for up to 30 days. Insulin secretion was studied after oral and intravenous administration of glucose and glucagon secretion after intravenous arginine. Islet morphology was examined and insulin secretion and glucose oxidation were measured in isolated islets. RESULTS: Fasting plasma glucose levels were reduced by GRA (6.0 +/- 0.2 vs 7.4 +/- 0.5 mmol/l; p = 0.017). The acute insulin response to intravenous glucose was augmented (1,300 +/- 110 vs 790 +/- 64 pmol/l; p < 0.001). The early insulin response to oral glucose was reduced in mice on HFD + GRA (1,890 +/- 160 vs 3,040 +/- 420 pmol/l; p = 0.012), but glucose excursions were improved. Intravenous arginine significantly increased the acute glucagon response (129 +/- 12 vs 36 +/- 6 ng/l in controls; p < 0.01), notably without affecting plasma glucose. GRA caused a modest increase in alpha cell mass, while beta cell mass was similar to that in mice on HFD + vehicle. Isolated islets displayed improved glucose-stimulated insulin secretion after GRA treatment (0.061 +/- 0.007 vs 0.030 +/- 0.004 pmol islet(-1) h(-1) at 16.7 mmol/l glucose; p < 0.001), without affecting islet glucose oxidation. CONCLUSIONS/INTERPRETATION: Chronic glucagon receptor antagonism in HFD-fed mice improves islet sensitivity to glucose and increases insulin secretion, suggesting improvement of key defects underlying impaired glucose tolerance and type 2 diabetes.

Evaluation of sentinel node identification with isosulfan blue in gastric cancer.

AIMS: The aim of this study was to clarify whether the metastasis of gastric cancer can be detected by identifying the sentinel lymph nodes (SNs) using only lymphatic dye. METHOD: The study was based on 101 patients clinically diagnosed with T1 and T2 gastric cancer. Isosulfan blue was intraoperatively injected within the submucosal layer around the lesion through an endoscope and blue stained nodes (BNs) were identified as SNs and harvested. Standard radical gastrectomy with D2 lymphadenectomy was performed on all patients and SNs and other nodes were evaluated. RESULTS: Out of 101 patients, 21 had lymph-node metastases. The accuracy of SN identification was 97.0% (98/101) and the metastasis detection rate was 85.7% (18/21). With improvement of the manoeuvre in the latter 84 cases, the detection rate was elevated up to 100.0% (18/18), as was the accuracy (84/84). CONCLUSION: The sentinel concept using lymphatic dye is applicable to gastric cancer, but the identification of SNs with lymphatic dye requires some practice. Furthermore, the issue of intraoperative metastasis detection remains to be resolved.

Molecular analysis of age-related changes of Streptococcus anginosus group and Streptococcus mitis in saliva.

The purpose of this study was to survey the prevalence of streptococcal species, especially Streptococcus anginosus (which has been reported to be associated with cancer in the upper digestive tract), Streptococcus constellatus, and Streptococcus intermedius in the saliva of different age groups. A sequence analysis of 16S rDNA was performed and DNA quantified using real-time polymerase chain reaction. The S. anginosus level increased with age, whereas the levels of S. constellatus and S. intermedius did not change. Streptococcus mitis was the predominant species in the saliva of all the age groups but, unlike the S. anginosus, the proportion of S. mitis in the salivary bacteria decreased with age. The increase in S. anginosus with age should be carefully monitored because of its association with diseases, including cancer.

Molecular evolution and diversification of snake toxin genes, revealed by analysis of intron sequences.

The genes encoding erabutoxin (short chain neurotoxin) isoforms (Ea, Eb, and Ec), LsIII (long chain neurotoxin) and a novel long chain neurotoxin pseudogene were cloned from a Laticauda semifasciata genomic library. Short and long chain neurotoxin genes were also cloned from the genome of Laticauda laticaudata, a closely related species of L. semifasciata, by PCR. A putative matrix attached region (MAR) sequence was found in the intron I of the LsIII gene. Comparative analysis of 11 structurally relevant snake toxin genes (three-finger-structure toxins) revealed the molecular evolution of these toxins. Three-finger-structure toxin genes diverged from a common ancestor through two types of evolutionary pathways (long and short types), early in the course of evolution. At a later stage of evolution in each gene, the accumulation of mutations in the exons, especially exon II, by accelerated evolution may have caused the increased diversification in their functions. It was also revealed that the putative MAR sequence found in the LsIII gene was integrated into the gene after the species-level divergence.

Lower blood glucose, hyperglucagonemia, and pancreatic alpha cell hyperplasia in glucagon receptor knockout mice.

Glucagon, the counter-regulatory hormone to insulin, is secreted from pancreatic alpha cells in response to low blood glucose. To examine the role of glucagon in glucose homeostasis, mice were generated with a null mutation of the glucagon receptor (Gcgr(-/-)). These mice display lower blood glucose levels throughout the day and improved glucose tolerance but similar insulin levels compared with control animals. Gcgr(-/-) mice displayed supraphysiological glucagon levels associated with postnatal enlargement of the pancreas and hyperplasia of islets due predominantly to alpha cell, and to a lesser extent, delta cell proliferation. In addition, increased proglucagon expression and processing resulted in increased pancreatic glucogen-like peptide 1 (GLP-1) (1-37) and GLP-1 amide (1-36 amide) content and a 3- to 10-fold increase in circulating GLP-1 amide. Gcgr(-/-) mice also displayed reduced adiposity and leptin levels but normal body weight, food intake, and energy expenditure. These data indicate that glucagon is essential for maintenance of normal glycemia and postnatal regulation of islet and alpha and delta cell numbers. Furthermore, the lean phenotype of Gcgr(-/-) mice suggests glucagon action may be involved in the regulation of whole body composition.

[Recurrence of bullous pemphigoid after surgery: report of a case].

We report a 38-year-old woman with myoma uteri and bullous pemphigoid controlled by oral prednisolone (7.5 mg.day-1). She underwent transabdominal hysterectomy under epidural anesthesia using mepivacaine supplemented with intravenous midazolam and butorphanol, without untoward event. On the postoperative day 2, recurrence of bullous pemphigoid was noted. The skin lesion of pemphigoid was improved after increasing the prednisolone dose up to 60 mg a day.

Diagnostic value of pulsed Doppler echocardiography in acute pulmonary thromboembolism--comparison with pulmonary angiography and pulmonary artery pressure.

The ratio of acceleration time to right ventricular ejection time (AcT/RVET) can be derived from the blood flow patterns recorded by pulsed wave Doppler echocardiography. In chronic cor pulmonale, AcT/RVET negatively correlates with pulmonary artery pressure (PAP). The present study evaluated the diagnostic value of AcT/RVET by comparing this variable with indices derived from pulmonary angiography (PAG) and PAP in 16 patients with acute pulmonary thromboembolism (APTE). AcT/RVET, PAP, and PAG severity indices (Miller index (MI) and UPET objective angiographic index (UI)) were measured during the acute phase on admission and the chronic phase after treatment. In the acute phase, AcT/RVET correlated with mean PAP (mPAP) (r=-0.68, p<0.05) and total pulmonary resistance (TPR) (r=-0.66, p<0.05), but not with MI or UI. During the chronic phase, AcT/RVET did not correlate with mPAP or TPR, but with both PAG indices (MI: r=0.76, p<0.05, UI: r=0.65, p<0.05). Before treatment of the APTE, AcT/RVET remained at low levels and could be used as an index of pulmonary hypertension. After treatment, however, following improvement of PAP, AcT/RVET was not useful for evaluating PAP, but might serve as an index for evaluating the volume of residual thrombi.

[No relation between angiotensin-converting enzyme (ACE) inhibitor-induced cough and ACE gene polymorphism, plasma bradykinin, substance P and ACE inhibitor concentration in Japanese patients].

Persistent dry cough is well known as the most common side-effect of angiotensin-converting enzyme (ACE) inhibitors. We examined the relationship between a cough and ACE gene polymorphism, plasma bradykinin (BK), substance P (SP) and ACE inhibitor concentrations in patients with hypertension or chronic nephritis. First, ACE genotyping was carried out in 96 patients, 42 with coughs and 54 without coughs, which had been treated with various kinds of ACE inhibitors. However, no significant difference in the ACE genotypes was observed between the two groups. Second, the plasma concentrations of BK, SP and ACE inhibitor were measured in 12 patients, which were treated with trandolapril at a daily dose of 1 mg for 4-33 weeks. In 3 patients, the cough was induced during the trandolapril therapy, while it was induced not in 9 patients. The plasma levels of BK and SP did not significantly change after trandolapril administration in the patients with and without coughs. Between the two groups, there were no significant differences in the plasma levels of BK and SP either before or after the trandolapril therapy. Also the plasma concentrations of trandolapril and trandolaprilat, the active metabolite of trandolapril, did not significantly differ between the two groups. These results suggest that there is no significant relationship between the ACE inhibitor-induced cough and ACE gene polymorphism, plasma BK, SP and ACE inhibitor concentrations in patients with hypertension or chronic nephritis.

Surface analysis of Ti-15Zr-4Nb-4Ta alloy after implantation in rat tibia.

A new Ti-15Zr-4Nb4Ta alloy without V was implanted in rat tibiae for 6-48 weeks. The new bone formation surrounding the Ti implant in bone marrow, surface analysis of the Ti alloy after implantation, and metal concentrations in dried bone tissue containing new bone were investigated. New bone was well formed around the Ti-15Zr-4Nb-4Ta alloy implanted in bone marrow. The mean thickness of the new bone increased up to 24 weeks after implantation, and changes in the mean thickness thereafter, up to 48 weeks. were very small. The number of corrosion pits observed in the Ti-6Al-4V extra low interstitial (ELI) alloy surface tended to be slightly more than that of the Ti-5Zr-4Nb-4Ta alloy implant. The concentrations of metal elements in the bone tissue containing the new bone tended to increase slightly more than in bones without the implant.

Availability of oncogene activated production system for mass production of light chain of human antibody in CHO cells.

We previously established a ras-oncogene amplified Chinesehamster ovary (CHO) cell line, named ras clone I, as anuniversal host cell line for oncogene activated production(OAP) system to mass-produce recombinant protein by activationof the cytomegalovirus immediate early (CMV) promoter with ras protein. The lambda light chain(C5lambda) of human monoclonal antibody HB4C5 is expected tobe potentially useful for lung cancer targeting. We generated aC5lambda hyper-producing cell line by transfecting ras cloneI with the C5lambda gene expression plasmid regulated by theCMV promoter, of which productivity was 5.3 times greater thanthe hyper productive CHO cell line generated by using conventional CHO cells. Introduction of the adenovirus E1A geneinto the hyper-producing cell line derived from ras clone I resulted in further 9.5 times enhancement of the productivity,suggesting the synergistic effect of E1A and ras oncogenes on the recombinant protein production driven by the CMV promoter. In addition, intracellular accumulation of C5lambda andupregulation of BiP was found in hyper-producing cell lineswhich were introduced E1A and ras oncogene. This resultsuggests that excessive intracellular accumulation ofC5lambda protein, which might be caused by that the amount of produced C5lambda in ER is beyond the ability of CHO cells to secrete, might signal the BiP promoter. Our data imply that ras clone I is available as a general host cell for establishing the recombinant protein hyper-producing CHOcells by the OAP system, and suggest that further mass production of recombinant proteins in the OAP system can be possible by clarifying the accurate role of upregulated BiP protein.

Review: melanocyte migration and survival controlled by SCF/c-kit expression.

Melanocytes are derived from neural crest and migrate along the dorsolateral pathway to colonize the final destination in the skin. Stem cell factor and its receptor c-kit were identified as gene products of Sl and W mutant loci; both of them were known to have defects in melanocytes survival. In this review, we focus on the function of stem cell factor and c-kit in melanocyte migration and survival, which has become clearer in the last decade. By analysis of both molecules in wild-type and white spotting mutant mice, ligand and receptor set were shown to play multiple roles in the development of melanocytes in mouse ontogeny. Functional blockade of c-kit by specific monoclonal antibody illustrated distinct c-kit dependent and independent stages in melanocyte development. Finally, SCF transgene expression demonstrated that part of the c-kit dependent step is regulated by spatiotemporally specific ligand expression and also indicated the presence of c-kit independent melanocyte stem cells in postnatal skin.

Left ventricular mass and global function in essential hypertension after antihypertensive therapy.

This study compares left ventricular global function in 55 patients (25 with hypertrophy and 30 without hypertrophy) with essential hypertension, whose blood pressure had been stable for longer than 1 year, with that in 35 healthy subjects. Left ventricular global function was calculated using the echocardiographic Doppler index as described by Tei et al. (Tei index). No significant differences were observed in echocardiographic left ventricular systolic function between the three groups. Left ventricular global function differed significantly between the three groups, however, suggesting impaired left ventricular global function even in the absence of left ventricular hypertrophy. Similar changes were observed in the ratio of peak velocity of atrial systole to peak velocity of early diastole during left ventricular inflow waveform (A/E), indicating that this disorder mainly reflects impaired left ventricular diastolic function. Unlike A/E, the Tei index is not affected by increasing age, and, therefore, is appropriate for evaluating left ventricular global function in hypertensive patients, most of whom are middle-aged or older. This index may be useful for determining treatment strategy and evaluating treatment effects.

Dipeptidyl peptidase IV (DPIV/CD26) degradation of glucagon. Characterization of glucagon degradation products and DPIV-resistant analogs.

Over the past decade, numerous studies have been targeted at defining structure-activity relationships of glucagon. Recently, we have found that glucagon(1-29) is hydrolyzed by dipeptidyl peptidase IV (DPIV) to produce glucagon(3-29) and glucagon(5-29); in human serum, [pyroglutamyl (pGlu)(3)]glucagon(3-29) is formed from glucagon(3-29), and this prevents further hydrolysis of glucagon by DPIV (H.-U. Demuth, K. Glund, U. Heiser, J. Pospisilik, S. Hinke, T. Hoffmann, F. Rosche, D. Schlenzig, M. Wermann, C. McIntosh, and R. Pederson, manuscript in preparation). In the current study, the biological activity of these peptides was examined in vitro. The amino-terminally truncated peptides all behaved as partial agonists in cyclic AMP stimulation assays, with Chinese hamster ovary K1 cells overexpressing the human glucagon receptor (potency: glucagon(1-29) > [pGlu(3)]glu- cagon(3-29) > glucagon(3-29) > glucagon(5-29) > [Glu(9)]glu- cagon(2-29)). In competition binding experiments, [pGlu(3)]glucagon(3-29) and glucagon(5-29) both demonstrated 5-fold lower affinity for the receptor than glucagon(1-29), whereas glucagon(3-29) exhibited 18-fold lower affinity. Of the peptides tested, only glucagon(5-29) showed antagonist activity, and this was weak compared with the classical glucagon antagonist, [Glu(9)]glucagon(2-29). Hence, DPIV hydrolysis of glucagon yields low affinity agonists of the glucagon receptor. As a corollary to evidence indicating that DPIV degrades glucagon (Demuth, et al., manuscript in preparation), DPIV-resistant analogs were synthesized. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to assess DPIV resistance, and it allowed kinetic analysis of degradation. Of several analogs generated, only [D-Ser(2)] and [Gly(2)]glucagon retained high affinity binding and biological potency, similar to native glucagon in vitro. [D-Ser(2)]Glucagon exhibited enhanced hyperglycemic activity in a bioassay, whereas [Gly(2)]glucagon was not completely resistant to DPIV degradation.

Regulation of E- and P-cadherin expression correlated with melanocyte migration and diversification.

Melanocytes (Mc) and their progenitors melanoblasts (Mb) are derived from the neural crest and migrate along the dorsolateral pathway to colonize the dermis, the epidermis, and finally the hair matrix. To examine the involvement of cadherins in the migration of Mc lineage cells, we combined flow cytometric analysis of dissociated live cells with immunohistochemical staining of tissue sections to quantify the level of cadherin expression on the surface of Mb/Mc. At 11.5 days postcoitum, Mb are in the dermis and are E-cadherin(-)P-cadherin(-) (E-cad(-)P-cad(-)). During the next 48 h, a 200-fold increase of E-cadherin expression is induced on the surface of Mb prior to their entry into the epidermis, thereby forming a homogeneous E-cad(high)P-cad(-/low) population. The cadherin expression pattern then diversifies, giving rise to three populations, an E-cad(-)P-cad(-) dermal population, E-cad(high)P-cad(low) epidermal population, and E-cad(-)P-cad(med-high) follicular population. In all three populations, the patterns of expression are region-specific, being identical with those of surrounding cells such as keratinocytes and fibroblasts, and are preserved before and after pigmentation. While most of the epidermal Mb/Mc disappear after the neonatal stage in normal mice, forced expression of steel factor in the epidermis of transgenic mice promotes survival of epidermal Mb/Mc, maintaining epidermal-type cadherin expression pattern (E-cad(high)P-cad(low)) throughout the postnatal life. These findings indicate the involvement of extrinsic cues in coordinating the cadherin expression pattern of Mb/Mc and suggest a role for E- and P-cadherins in guiding Mc progenitors to their final destinations.

Evaluation of right-ventricular function by Doppler echocardiography in patients with chronic respiratory failure.

In patients with chronic respiratory failure, right-ventricular function was non-invasively evaluated by Doppler echocardiography. A total of 31 patients (16 men, 15 women; mean age 65.8 +/- 7.12 years) with pulmonary tuberculosis sequelae who had received home oxygen therapy during the preceding 6 or more months, were studied. Right-ventricular inflow and outflow waveforms were recorded, and right-ventricular function was evaluated using a new Doppler index combining systolic and diastolic function. On continuous wave Doppler echocardiography, estimated systolic pulmonary arterial pressure was calculated from the gradient between the right atrium and right ventricle. There was no correlation between the new Doppler index and systolic pulmonary arterial pressure, and some patients showed high index values despite low systolic pulmonary arterial pressure. The new Doppler index facilitated evaluation of ventricular function irrespective of right-ventricular afterload.

Recombinant light chain of human monoclonal antibody HB4C5 as a potentially useful lung cancer-targeting vehicle.

Recombinant lambda light chain of lung cancer-reacting human monoclonal antibody HB4C5 was expressed in Escherichia coli. Expression in bacteria ensured the generation of homogeneous light chain species devoid of activity-hampering N-linked glycosylation usually found in the light chain CDR-1 of HB4C5. Molecular engineering was also employed to eliminate the C-terminal two amino acid residues, i.e., Cys and Ser, to prevent the formation of lambda light chain dimers which are less reactive than the monomeric form. The lambda light chain was overexpressed in E. coli as inclusion bodies, which were solubilized, refolded, and treated with Aeromonas proteolytica aminopeptidase to remove the N-terminal Met with subsequent natural cyclization of the penultimate Gln residue to pyroglutamate, the same N-terminal end as that of naturally occurring lambda light chain in HB4C5. Monomeric recombinant lambda light chains, both before and after removal of the N-terminal Met residue, were 40 times more immunoreactive than the parent HB4C5. The immunostaining of lung cancer tissue sections with the recombinant lambda light chain indicated cancer-specific reactions to all specimens of adenocarcinoma, squamous cell carcinoma and large cell carcinoma histologies, but did not react with small cell carcinoma. Tumor radioimmunoimaging experiments in LC6 (lung squamous cell carcinoma line)--xenografted nude mice by the i.p. injection of 125I-labeled recombinant lambda light chain and 125I-labeled human lambda light chain control gave tumor-specific and recombinant lambda light chain-dependent images on day 5 postinjection, and images were also detectable on day 3. Biodistribution studies with 125I-labeled recombinant lambda light chain demonstrated that the lambda light chain could penetrate better into the tumor sites, both at the necrotic and solid parts of the xenograft, as compared to our previous results with 125I-labeled HB4C5 which could localize to the necrotic part only. These results suggest that the recombinant lambda light chain is potentially useful as a lung cancer-targeting vehicle, for such as radioimmunoimaging and radioimmunotherapy, with least possible adverse immunogenic effects.