Effect of administering activated lymphocytes originated from the dam on the immune cell reaction in Holstein calves.

Injection of lymphokine activated killer (LAK) cells is known as useful for activation of cellular immune system. Although the effect of LAK cells has been clarified in human or mice, this effect on function of immune cells has not been examined in calves. Healthy ten Holstein calves were injected with the LAK cells 2 days after birth (LAK Group), and another eight calves were observed as controls (Control Group). All calves received the colostrum formulation on the day of birth, and then, were inoculated with a live attenuated vaccine of bovine herpesvirus (BHV)-1 at 2 (the first vaccination) and 6 (the second vaccination) weeks after birth. Peripheral blood of their dam obtained 3 weeks before calving was used for preparation of LAK cells. Blood samples were taken prior to vaccine inoculation and 3 days after the first inoculation, as well as 3 and 6 days after the second vaccination from all calves. Numbers of CD8+ and CD21+ cells increased significantly after the second vaccination in the LAK Group compared with Control Group. The present study suggested the improved effect of injecting LAK cells originated from dams on immune cells function of young calves after BHV-1 live vaccine.

Effects of rotational acetabular osteotomy on the mechanical stress within the hip joint in patients with developmental dysplasia of the hip: a subject-specific finite element analysis.

In this study we used subject-specific finite element analysis to investigate the mechanical effects of rotational acetabular osteotomy (RAO) on the hip joint and analysed the correlation between various radiological measurements and mechanical stress in the hip joint. We evaluated 13 hips in 12 patients (two men and ten women, mean age at surgery 32.0 years; 19 to 46) with developmental dysplasia of the hip (DDH) who were treated by RAO. Subject-specific finite element models were constructed from CT data. The centre-edge (CE) angle, acetabular head index (AHI), acetabular angle and acetabular roof angle (ARA) were measured on anteroposterior pelvic radiographs taken before and after RAO. The relationship between equivalent stress in the hip joint and radiological measurements was analysed. The equivalent stress in the acetabulum decreased from 4.1 MPa (2.7 to 6.5) pre-operatively to 2.8 MPa (1.8 to 3.6) post-operatively (p < 0.01). There was a moderate correlation between equivalent stress in the acetabulum and the radiological measurements: CE angle (R = -0.645, p < 0.01); AHI (R = -0.603, p < 0.01); acetabular angle (R = 0.484, p = 0.02); and ARA (R = 0.572, p < 0.01). The equivalent stress in the acetabulum of patients with DDH decreased after RAO. Correction of the CE angle, AHI and ARA was considered to be important in reducing the mechanical stress in the hip joint.

Molecular characterization of fungal populations on the tongue dorsum of institutionalized elderly adults.

OBJECTIVES: To characterize the global composition of oral fungal populations in frail elderly adults and to investigate the relationship with their health status. MATERIALS AND METHODS: We investigated the fungal populations on the tongue dorsum in 291 institutionalized elderly adults by molecular PCR-based techniques using internal transcribed spacer regions of nuclear ribosomal DNA. RESULTS: Quantitative PCR analysis showed that fungi were present on the tongue dorsum of 128 subjects at ≥10(4) CFU per sample, and 35 of them exceeded 10(5) CFU per sample. Length heterogeneity-PCR analysis and nucleotide sequence determinations showed that Candida albicans was most frequently detected in those subjects with fungi at ≥10(4) CFU per sample (105 subjects), followed by Candida dubliniensis (78), Malassezia restricta (57), and Candida tropicalis (45). Statistical analysis revealed that those subjects with ≥10(5) CFU of fungi other than C. albicans per sample had an increased risk of fever (≥7 febrile days per 12 months) compared with subjects with <10(5) CFU per sample, after adjustment for other fever-associated confounding factors. CONCLUSIONS: These data demonstrate that the oral cavity of the elderly is inhabited by a diverse array of fungi not limited to typical Candida species and they suggest that the diversity in distribution is associated with health status.

Surgery for clavicular and humeral fractures in an osteopetrotic patient: a case report.

Osteopetrosis is a rare disease characterised by generalised sclerosis of the bone. Surgical treatment for fractures in osteopetrotic bones is difficult due to their hardness. We report successful surgical treatment of humeral and clavicular fractures in a 30-year-old osteopetrotic patient with severe multiple trauma. Two years after surgery, the patient had a full range of movement at the shoulder and elbow, with good bone union and alignment.

Nestin expression in the kidney with an obstructed ureter.

Nestin is an intermediate filament protein originally identified in neuroepithelial stem cells. This cytoskeletal-associated protein is also expressed in some non-neuronal organs including renal tubular cells and glomerular endothelial cells during kidney development. Little is known, however, about nestin expression in the kidney during injury. In this study, we find nestin expression induced in renal tubular and interstitial myofibroblasts in the adult rat kidney following unilateral ureteral obstruction. The degree of nestin expression was well correlated with the degree of tubulointerstitial fibrosis. Immunohistochemical identification of specific nephron segments showed that nestin was primarily expressed by proximal tubules, partially by distal tubules and thick ascending limbs of Henle but not by collecting ducts. The nestin-positive tubular cells also expressed vimentin and heat-shock protein 47 (HSP47) suggesting these cells reverted to a mesenchymal phenotype. Not all vimentin- or HSP-expressing cells expressed nestin; however, suggesting that nestin is distinct from these conventional mesenchymal markers. Nestin expression was also found associated with phenotypical changes in cultured renal cells induced by hypoxia or transforming growth factor-beta. Nestin expression was located in hypoxic regions of the kidney with an obstructed ureter. Our results indicate that nestin could be a novel marker for tubulointerstitial injury.

Tyrosinase induction and inactivation in normal cultured human melanocytes by endothelin-1.

Since endothelin was found to be expressed in epithelial cells as well as in vascular endothelial cells, the functional regulation of melanocytes with endothelin has been actively investigated. In particular, it has been suggested that endothelin may influence pigmentation and depigmentation, which are mediated by melanocytes. In the present study, we investigated the regulation of melanocyte function and tyrosinase expression by endothelin from the point of view of tyrosinase protein expression and enzyme activity. The influence of endothelins on melanocyte function was assessed. Melanocytes showed a dose-dependent increase in cell proliferation with the addition of endothelin-1. When the confluence of melanocytes was cultured with endothelin-1 for 72 h, tyrosinase activity in melanocytes was significantly and dose-dependently decreased. In contrast, there was no significant change with endothelin-3. However, tyrosinase protein expression of melanocytes was significantly and dose-dependently increased by endothelin-1, but endothelin-3 had no effect. Both the suppression of enzyme activity and the enhanced protein expression were regulated by the ETA receptor antagonist, BQ123. In view of these observations, we conclude that endothelin-1-induced tyrosinase is mediated by ETA receptors. However, the reason for the decrease in the specific activity of tyrosinase remains unknown, and our results suggest that another mechanism underlying the activation of tyrosinase is present in addition to the inductive action of endothelin-1 on tyrosinase.

Widespread crystalline inclusions affecting podocytes, tubular cells and interstitial histiocytes in the myeloma kidney.

Numerous crystalline inclusions were observed in glomerular and tubular epithelial cells in a 46-year-old female patient with multiple myeloma and renal dysfunction. On light microscopy, epithelial cells were filled with homogenous materials and were remarkably swollen. Infiltrations of histiocytes with expanded cytoplasm were also seen in the interstitium of the kidney and bone marrow. On electron microscopy, cytoplasmic inclusions had crystalline structure showing rhomboid and oval shapes. Immunofluorescence study revealed that these cells were positive for IgG-kappa. The combination chemotherapy followed by autologous stem cell transplantation led to a partial resolution of her renal dysfunction, continued by a slight reduction in the number of crystalline-containing podocytes at the second renal biopsy. Crystal inclusions in the kidney are rarely found and cause renal impairment in multiple myeloma.

Chemical cross-linking of peptides derived from RecA with single-stranded oligonucleotides containing 5-formyl-2'-deoxyuridine.

We report the first example of chemical cross-linking of 5-formyl-2'-deoxyuridine containing oligonucleotides with oligopeptides through a Schiff base formation. Twenty amino acid residue peptides investigated here were derived from the DNA binding site of RecA protein. We have demonstrated that the lysine residue placed at the 6th or 8th position from the N-terminus of the peptide directly contacts with DNA.

Alpha-tocopherol affects the urinary and biliary excretion of 2,7,8-trimethyl-2 (2'-carboxyethyl)-6-hydroxychroman, gamma-tocopherol metabolite, in rats.

In this study, we investigated a change in the excretory content of 2,7,8-trimethyl-2(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC), a gamma-tocopherol (gamma-Toc) metabolite, in rat urine and bile by using a new high-performance liquid chromatography-electrochemical detection (HPLC-ECD) method. In this determination, CEHC [alpha- and gamma-CEHC, where alpha-CEHC = 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman] in the biological specimens were treated with 3 N methanolic HCl to hydrolyze conjugates and to promote esterification. The methylated samples were extracted by n-hexane/water (1:2). The analyses of the methyl esters of alpha-CEHC and gamma-CEHC were performed by an HPLC-ECD using an ODS-3 column at 35 degrees C. The mobile phase was acetonitrile/water (45:55, vol/vol) containing 50 mM sodium perchlorate. After rat urine and bile samples, respectively, were methylated as described above, methylated biliary metabolites were identified by liquid chromatography-mass spectrometry as methyl esters of gamma-CEHC. Furthermore, we examined the differences in the excretion of gamma-CEHC between rat urine and bile after an oral administration of gamma-Toc or alpha- + gamma-Toc by the above HPLC method. In the gamma-Toc group, each vitamin E-deficient rat was given 0.5 mL of a stripped corn oil preparation containing 10 mg of gamma-Toc. In the alpha- + gamma-Toc group, the rat was given 10 mg of alpha-Toc and 10 mg of gamma-Toc. The content of gamma-CEHC in rat urine from the alpha- + gamma-Toc group was increased more in comparison to the gamma-Toc group at 18-36 h after oral administration. Moreover, the content of gamma-CEHC in rat bile in the alpha- + gamma-Toc group was increased more in comparison to the gamma-Toc group at 6-18 h after oral administration. Therefore, we have suggested that gamma-CEHC was shifted mainly to urinary excretion after gamma-CEHC had been excreted into the bile. Furthermore, we assume that alpha-Toc may affect the metabolism of gamma-Toc to gamma-CEHC in the body.

A novel traceless resin-bound guanidinylating reagent for secondary amines to prepare N,N-disubstituted guanidines.

[reaction: see text]. We report the development of a solid support-linked guanidinylating reagent. This reagent consists of a urethane-protected triflyl guanidine attached to the resin via a carbamate linker. It allows for rapid synthesis of guanidines from a variety of amines. It provides access to N-alkyl/aryl- or N,N-dialkylguanidines under mild conditions. Cleavage with 50% TFA produces target molecules in high yields and purity. The ability to guanidinylate secondary amines is a significant feature of this guanidinylating reagent.

[Troubles inherent to long-term home parenteral nutrition (HPN) and the use of technologies to improve communication between medical institutions and patients].

The continuous advance of technology has contributed to the improvement of the quality of life of patients under long-term home parenteral nutrition (HPN). But the longer the patient stays home, the higher the risk of many kinds of troubles. We designed an HPN system in June 1999, which uses E-mail for patients and to contact with each other (including the patient's family) and to prevent troubles from worsening. We describe the case of a patient which shows the efficacy and usefulness of our system, which makes use of the Internet, video mail, voice mail and electric medical records.

Molecular cloning and expression of aminopeptidase A isoforms from rat hippocampus.

The full-length cDNA encoding aminopeptidase A (APAL) was cloned from a rat hippocampus cDNA library. A short variant aminopeptidase A (APAS), produced by deletion, was also cloned. In the case of APAL, the longest open reading frame encodes 945 amino acid residues with a calculated molecular mass of 108 kDa, and the deduced amino acid sequence shows 76, 86 and 78% identity with its human, murine and porcine counterparts, respectively. Rat aminopeptidase A mRNAs were detected in the kidney, liver, heart and brain by Northern blot analysis. When overexpressed in COS-1 cells, APAL shows apparent aminopeptidase A activity, whereas APAS does not.

Impaired Kupffer cell function and effect of immunotherapy in obstructive jaundice.

BACKGROUND: Obstructive jaundice is frequently associated with septic complications. This study examined the influence of biliary obstruction on bacterial clearance and translocation. The study focused on the phagocytic and killing activities of Kupffer cells and the preventive effect on bacterial translocation of OK-432, which is a hemolytic streptococcal preparation developed as a biological response modifier. METHODS: To study the mechanism of sepsis in obstructive jaundice, two groups of Wistar rats were examined: rats subjected to common bile duct ligation (CBDL) and rats subjected to a sham operation. Bacterial clearance, organ distribution, hepatic blood flow, and phagocytic function of Kupffer cells were examined. To evaluate the effect of OK-432 on bacterial translocation, rats were divided into three groups: sham operation + phosphate-buffered saline (PBS), CBDL + PBS, and CBDL + OK-432. RESULTS: In this study, clearance of Escherichia coli. from the peripheral blood in CBDL rats was decreased significantly compared with that in sham-operated rats. Significant decreases in E.coli trapped in the liver and in hepatic blood flow were observed in CBDL rats compared with sham-operated rats. Phagocytic activity and superoxide production of Kupffer cells isolated from CBDL rats were significantly lower than in sham-operated rats. The incidence of bacterial translocation in CBDL rats was increased significantly, and oral administration of OK-432 prevented it. CONCLUSION: The results suggest that susceptibility to infection in obstructive jaundice is due to impaired phagocytic function of Kupffer cells. Furthermore, obstructive jaundice promotes bacterial translocation, and OK-432 may be useful in preventing this translocation.

Interaction of peptides derived from RecA with single-stranded oligonucleotides containing 5-formyl-2'-deoxyuridine.

We report the first example of chemical cross-linking of 5-formyl-2'-deoxyuridine containing oligonucleotides with oligopeptides through a Schiff base formation. Twenty amino acid residue peptides investigated here were derived from the DNA binding site of RecA protein. We have demonstrated that the lysine residue placed at the 6th or 8th position from the N-terminus of the peptide directly contacts with DNA.

Surviving and proliferating faculty of fibroblasts cultured under no-serum and poor nutritional conditions. The basic study on hybridation of cultured cells with an artificial matrix.

BACKGROUND: In this study, we measured survival periods of fibroblasts cultured under conditions providing minimal nutrition. This investigation aims to develop the high affinity hybrid artificial organs on tissues or organs finally. METHODS: Dermal fibroblasts were inoculated on 96-well multiplate according to routine technique. The medium of these cells maintained for 24 hours replaced the new DME medium containing 10% FBS (group 1) or without FBS (group 2) and then cultured for 10 days without medium change. The surviving cell numbers were measured using MTS assay. RESULTS: Surviving cell numbers of both groups decreased progressively during the 10-day assay period. Overall 25-40% of the fibroblasts survived for at least 10 days. It decreased significantly, however, in group 2 from day 2 through day 4 as compared to group 1. In contrast, the cell number decreased in group 1 to a greater extent than in group 2 after day 6. When the cells that survived until day 10 were then cultured with fresh DME medium containing 10% FBS for 3 more days, the cell numbers returned to 50-100% of the initial level CONCLUSIONS: These results suggest that fibroblasts cell function is maintained under poor nutritional conditions, i.e., without serum, possibly via an autocrine or paracrine mechanism. These data could contribute much to the research of hybrid artificial organs, including an artificial trachea, in which hybridization of cultured cells with artificial matrices remains to be a basic process, and has to serve in bloodless postimplantation period.

Interaction between antioxidants and hydroquinone/bisphenol.

We have investigated the stability of semiquinone radical (SQ .) produced from the mixture of hydroquinone (HQ) and bisphenol A (BPA) in the presence of various antioxidants, some of which induce apoptosis in tumor cell lines. BPA produced no detectable amount of ESR signal. The acceleration of HQ oxidation by BPA results in the production of SQ .. The BPA-enhanced SQ . production may occur through either SQ. regeneration by electron transfer from BPA working as an antioxidant or the O2- scavenging activity of BPA. SQ . intensity of HQ/BPA was reduced by lower concentrations of sodium ascorbate, epigallocatechin, gallate and quercetin, but significantly enhanced by gallic acid. The initial SQ . was replaced by the radicals of antioxidants except lignin and salicylic acid, at higher concentrations, due to their radical-radical coupling or dismutation process. Among these compounds, gallic acid enhanced the radical intensity of SQ . to the greatest extent. The results suggest that antioxidants from foods suppress the activity of xenobiotics such as HQ, however, they produce their own radical at higher concentrations, suggesting their bimodal actions. The present study demonstrates the usefulness of ESR spectroscopy for the interaction between HQ/BPA and antioxidants.

Changes in endothelin-1, 6-keto-PG-F1 alpha, and TX-B2 in random pattern flaps.

In this study we investigated kinetic changes in the ratio of endothelin-1 (ET-1) and eicosanoids (6-keto-PG-F1 alpha TX-B2) in 20 x 60 mm random pattern flaps from rats. The ET-1 content of regions A (20 mm from the peripheral end) and B (20-40 mm) 6 h after surgery tended to decrease slightly compared to the ET-1 content immediately after surgery. The ET-1 content of region C (20 mm from the flap base) 6 h postoperatively increased significantly compared to that immediately after surgery. The ET-1 content of region C 6 h after surgery was significantly higher compared to that of regions A and B, which were obtained simultaneously. The ratios of eicosanoids in the three regions 6 h after surgery were significantly lower than those immediately after operation. However, the ratio in region A was higher than that in region C, showing that there was a difference in distribution in the flap between ET-1 and eicosanoids. The administration of an ETA receptor antagonist, FR-139317, extended the survival length of the flap. These results suggest that ET-1 can regulate the microcirculation in a flap directly and/or indirectly.

Differential effects of granulocyte/macrophage colony-stimulating factor (GM-CSF) in enhancing macrophage resistance to Legionella pneumophila vs Candida albicans.

It has been reported that granulocyte/macrophage colony-stimulating factor (GM-CSF), one of the hemopoietic growth factors which regulates the function of phagocytic cells, is a potent activator of cultured macrophages and induces antimicrobial activities as well as differentiation of precursor cells. In this study, we examined the ability of recombinant murine GM-CSF to activate mouse peritoneal macrophages to restrict the growth of two different microorganisms, Candida albicans and Legionella pneumophila, both of which are important opportunistic pathogens in an immunocompromised host. Treatment of thioglycollate-elicited BDF1 mouse macrophages with GM-CSF for 24 hr enhanced the anti-C. albicans activity of the macrophages in terms of inhibiting growth of the fungi. Reactive oxygen (H2O2) and IL-1 production by the macrophages were also enhanced by treatment with GM-CSF. However, no enhancement of anti-L. pneumophila activity of macrophages obtained from either susceptible A/J or resistant BDF1 mice to L. pneumophila infection after treatment with up to 1000 units/ml GM-CSF was observed under the same conditions. When the treatment time was extended to 72 hr. GM-CSF was still unable to induce anti-L. pneumophila activity. As a control study, treatment with recombinant IFN-gamma enhanced both anti-Candida and anti-Legionella activity in cultured macrophages under the same conditions used in the GM-CSF study. Measurement of cellular iron content revealed the low iron content in IFN-gamma-treated macrophages, but no decrease of iron in GM-CSF-treated macrophages compared with the control group, indicating a possible involvement of iron as a key factor in anti-L. pneumophila activity. Thus, the results of the study show that GM-CSF activation of elicited peritoneal macrophages is selective with regard to the type of antimicrobial activity induced.

Physiological roles of endothelium-derived nitric oxide in the epigastric island flaps of rabbits.

Nitric oxide (NO), identified as the mediator of endothelium-dependent relaxation of vascular smooth muscle, is known to cause a number of inflammatory conditions, especially in ischemia/reperfusion injury. This experimental study, using a rabbit epigastric island flap, was designed to investigate whether skin flap ischemia followed by reperfusion-influenced serum NO and c-GMP concentrations in the flap. In addition, we also investigated the premedicated effects of the NO synthase inhibitor and heparin on serum NO and c-GMP concentrations in skin flap ischemia/reperfusion. Serum NO concentration after 15, 30, 45, and 60 minutes of ischemia followed by reperfusion significantly increased compared with that in nonischemic control and elevated flaps. On the contrary, serum NO concentration was suppressed in L-NAME or aminoguanidine pretreated animals with ischemic group. Administration of heparin increased the serum NO concentration in elevated flaps, but suppressed it in ischemic flaps followed by reperfusion. The changes in serum c-GMP and NO concentrations were related in all of the experimental groups. These results suggest that NO may be derived from vascular endothelial cells and dilate peripheral vessels in compensation for ischemia.

Apoptosis induction resulting from proteasome inhibition.

Proteases are known to be involved in the apoptotic pathway. We report here that benzyloxycarbonyl (Z)-Leu-Leu-leucinal(ZLLLal), a leupeptin analogue, can induce apoptosis in MOLT-4 and L5178Y cells. ZLLLal is a cell-permeant inhibitor of proteasome. Among the protease inhibitors tested, only calpain inhibitor I (acetyl-Leu-Leu-norleucinal) and ZLLLal caused a marked induction of apoptosis in MOLT-4 cells. In contrast Z-Leu-leucinal, a specific inhibitor of calpain, did not induce apoptosis. When MOLT-4 cells were incubated in the presence of ZLLLal, p53 accumulated in the cells. These results strongly suggest that inhibition of proteasome induces p53-dependent apoptosis and that proteasome can protect cell from apoptosis.