Technical note: measurement of ferritin in bovine milk and its clinical significance

A quantitative ELISA was developed for bovine milk ferritin with an assay limit of 0.16 ng/mL of bovine spleen ferritin. Ferritin-binding activity was detected in bovine milk samples, and this binding activity was inhibited by increasing ionic strength with the addition of 0.5 M (NH₄)₂SO₄. Heat treatment (60°C, 20 min) of bovine milk in the presence of 0.5 M (NH₄)₂SO₄ resulted in a 15 to 58% increase in ferritin concentrations compared with untreated samples. Although the recovery of bovine spleen ferritin added to milk was still low (55 to 90%), even in the presence of increased ionic strength with 0.5 M (NH₄)₂SO₄, recovery was improved by heat treatment at 60°C for 20 min (92 to 95%). Milk ferritin concentrations in 30 milk samples from quarters of 25 cows with mastitis (mean ± SE: 134.2 ± 28.7 ng/mL) were significantly higher than those in 17 quarter milk samples from 17 noninfected lactating cows (7.2 ± 1.2 ng/mL), suggesting that bovine milk contains putative ferritin-binding proteins that inhibit immunoassay for milk ferritin and that bovine milk ferritin is an indicator of IMI.     

Time and temperature dependence of carbon/epoxy interface strength

Characterization of fiber/matrix interface is essential for the understanding of long-term properties of fiber reinforced composite materials. In this research, time and temperature dependence of carbon/epoxy interface strength were investigated. Unidirectional specimens were tested under tensile load up to failure, at various temperatures and testing speeds. The failure modes were identified as matrix dominant failure or interface dominant failure. A unit-cell model was considered to evaluate the stresses at the microscopic level and identify the critical points of highest stresses. Time and temperature dependent stress-concentration factor and thermal residual stress at the critical points were calculated using viscoelastic FEA. The micro stresses at the critical points were found to be properly represented by a bilinear curve with the interface dominant failure mode associated with the horizontal portion of the curve, suggesting that the interface strength is independent of time and temperature.     

Temporal and spatial expression of distinct troponin T genes in embryonic/larval tail striated muscle and adult body wall smooth muscle of ascidian

During development of the ascidian Halocynthia roretzi, the tadpole larva hatched from the tailbud embryo metamorphoses to the adult with a body wall muscle. Although the adult body wall muscle is morphologically nonsarcomeric smooth muscle, it contains a troponin complex consisting of three subunits (T, I, and C) as do vertebrate striated muscles. Different from vertebrate troponins, however, the smooth muscle troponin promotes actin-myosin interaction in the presence of high concentration of Ca2+, and this promoting property is attributable to troponin T. To address whether the embryonic/larval tail striated muscle and the adult smooth muscle utilize identical or different regulatory machinery, we cloned troponin T cDNAs from each cDNA library. The embryonic and the adult troponin Ts were encoded by distinct genes and shared only < 60% identity with each other. These isoforms were specifically expressed in the embryonic/larval tail striated muscle and the adult smooth muscle, respectively. These results may imply that these isoforms regulate actin-myosin interaction in different manners. The adult troponin T under forced expression in mouse fibroblasts was unexpectedly located in the nuclei. However, a truncated protein with a deletion including a cluster of basic amino acids colocalized with tropomyosin on actin filaments. Thus, complex formation with troponin I and C immediately after the synthesis is likely to be essential for the protein to properly localize on the thin filaments.     

Prevention of infections in a case with myelodysplastic syndrome by an intermittent subcutaneous administration of G-CSF

An 83-year-old male was admitted to our hospital because of pancytopenia and low grade fever on April 19, 1993. On admission, hematological data were as follows: WBC 1,000/microliters with 19% neutrophils, RBC 367 x 10(4)/microliters, Hb 9.5 g/dl and platelets 6.7 x 10(4)/microliters. Bone marrow examination revealed 6.6% myeloblasts and 33.5% erythroblasts. Morphological abnormalities included hypersegmentation, degranulation and pseudo-Pelger's nuclear anomaly in neutrophils. Based on these findings the diagnosis of refractory anemia with excess of blasts (RAEB) of the myelodysplastic syndrome (MDS) was made and therapy with low dose Cytarabine (Ara-C) was initiated in April 1993. The patient had two episodes of severe pneumonia in June and July. Therefore, 75 micrograms/day of G-CSF was given in addition to antibiotic therapy for the second episode of infection in July. Thereafter the severe infection subsided, and G-CSF administration was switched to an intermittent schedule (75 micrograms twice a week) since September. Cytarabine ocfosfate (100 mg/day) was added for 10-14 days at interval 1-2 months from October,1993. He has been well with no episode of infection for more than two year. One major concern regarding the clinical application of G-CSF in MDS patients is related to the possible stimulation of leukemic cell proliferation. Frequent hematological monitoring is necessary in patients with RAEB who are prone to develop acute myeloid leukemia. However, we administered G-CSF at a relatively low dose twice a week for over two year and could successfully prevent infections without inducing the leukemic changes.     

Iron nutrition and anaemia in a malaria-endemic environment: haematological investigation of the Gidra-speaking population in lowland Papua New Guinea

Blood examination was conducted for the four Gidra-speaking village groups in Papua New Guinea, who were characterized by high Fe intake and high malaria prevalence with marked inter-village differences. The northern riverine villagers, whose Fe intake was higher than the other three village groups, did not suffer from Fe-deficiency anaemia in their malaria-endemic environment; nor did the inland villagers, with their second highest Fe intake and their malaria-free environment, suffer from Fe-deficiency anaemia. However, several individuals of the southern riverine village suffered from anaemia in a malaria-endemic environment, although their Fe intake was almost the same as the inland villagers'. A considerable proportion of the coastal villagers were anaemic, reflecting the lowest Fe intake and the highest malaria prevalence. An inter-village comparison of the relationships between haemoglobin levels and transferrin saturation revealed that the southern riverine villagers needed smaller amounts of circulating Fe for erythropoiesis than the northern riverine and inland villagers, reflecting the long-term human-environment conditions such as the density of malaria vectors and the people's dietary habits. Fe supplementation was not judged effective against hypoferraemia and/or anaemia in such a population. As the incidence of malaria had no significant long-lasting effect on Fe stores or circulating Fe concentration, but did have an effect on anaemia, the hypothesis that malaria causes a transfer of Fe from the blood to parenchymal tissues as a defence against infectious diseases was not supported.     

The effect of TNP-470 on cell proliferation and urokinase-type plasminogen activator and its inhibitor in human lung cancer cell lines

The plasminogen activator system has been found to modulate neoplastic spread and angiogenesis in tumors. An angiogenesis inhibitor, TNP-470, has been shown to possess potent antitumor activities in various types of cancer cells. We therefore investigated the inhibitory effect of TNP-470 on cancer cell proliferation, and the suppressive effect of TNP-470 on urokinase-type plasminogen activator (u-PA) and its inhibitor (PAI-1), in human lung cancer cell lines in vitro. TNP-470 inhibited cell proliferation in a dose-dependent manner in both cell lines. u-PA and PAI-1 in culture supernatants were suppressed with the concentrations of TNP-470, in association with a decrease in viable cancer cells. Unchanged serum levels of u-PA and PAI-1 would be of great advantage to the diseased patients, since the plasminogen activator system has a crucial function in the process of distant metastasis.     

Detection of Salmonella enteritidis in shell and liquid eggs using enrichment and plating

Detection methods using various enrichment and plating media and immunoconcentration for Salmonella enteritidis in shell and liquid eggs were evaluated. For liquid egg samples naturally contaminated with S. enteritidis, pre-enrichment in 225 ml of buffered peptone water with cysteine followed by selective enrichment in 10 ml of tetrathionate broth was the superior, resulting in the detection of S. enteritidis in all samples on six of the seven types of selective agar substrate investigated. This enrichment procedure also enabled detection of S. enteritidis in most of artificially inoculated shell egg and pasteurized liquid egg samples.     

Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase delta and replication factor C

BACKGROUND: PCNA, an eukaryotic DNA sliding clamp interacts with replication factors and the cell cycle protein, p21(Cip1/Waf1) and functions as a molecular switch for DNA elongation. To understand how DNA replication is regulated through PCNA, elucidation of the precise mechanisms of these protein interactions is necessary. RESULTS: Loop-region mutants in which human PCNA sequences were substituted with the corresponding Saccharomyces cerevisiae PCNA regions were prepared. Analysis of their functions, along with previously prepared alanine scanning mutants, demonstrated that some loops interact with DNA polymerase delta (pol delta) and replication factor C (RFC). The p21 binding sites of PCNA, mapped by affinity measurement of the mutant forms, found to be located within a distinct structure of the PCNA monomer, overlap with RFC- and pol delta-interaction sites. Competition between p21 and pol delta or RFC for binding to PCNA results in efficient inhibition of its stimulation of pol delta DNA synthesis and RFC ATPase but not of PCNA loading on DNA by RFC. CONCLUSIONS: Semi-saturated amounts of p21 selectively block formation of the active pol delta complex but not the RFC-PCNA complex at 3'-ends of DNA primers. This differential effect may explain the specific inhibition of DNA replication by p21.     

A visual method for analysing bucco-lingual position of artificial posterior teeth. Part 1: use of the ridge crest

We have developed a visual analysis method to examine the spatial relationship between the edentulous ridges and the bucco-lingual position of the artificial posterior teeth in complete denture fabrication. In this system, a non-contact type shape measurement system is used. We applied this system to the plaster models and the wax dentures of an edentulous patient. Using the measurement data on the upper and lower plaster models, we reconstructed their shape three-dimensionally and determined the points regarded as the ridge crests and the inter-alveolar crest lines in the frontal sections. To generate the upper and lower ridge crest lines which consisted of the points of the ridge crests, interpolation by B-spline curves was applied. Furthermore, the loft surfaces that we regarded as the consecutive inter-alveolar crest lines, were generated between the upper and lower ridge crest lines. The surface models of the plaster models and artificial teeth were displayed on the cathode-ray tube display unit, and the ridge crest lines and the consecutive inter-alveolar crest lines were superimposed on the surface models. This method could be utilized to visualize and examine the bucco-lingual position of the artificial posterior teeth in reference to the inter-alveolar crest lines.     

Hsp40, a possible indicator for thermotolerance of murine tumour in vivo

The relationship between Hsp40/Hsp70 synthesis and the development of thermotolerance was investigated using mouse squamous cell carcinoma in vivo. To examine the thermotolerance, tumours were heated at 44 degrees C for 30 min as conditioning heating. After various intervals they were heated again at 44 degrees C for 90 min as challenge heating. The tumour response to heat was evaluated by the growth delay. Thermotolerance rapidly developed with increasing interval and reached a maximum at 12 h interval. Subsequently, thermotolerance gradually decayed and almost disappeared at 120 h interval. Under this condition, synthesis of Hsp40/Hsp70 increased after conditioning heating, reached a maximum at 12 h interval, then gradually decreased thereafter within 120 h. The kinetics of accumulation and decay of both Hsp40 and Hsp70 were very similar. The extent of thermotolerance was well correlated with the relative amount of Hsp40/Hsp70. These results obtained in vivo were very similar to those in vitro (Kaneko et al. 1995). Our findings suggest that Hsp40 could be a useful indicator of the degree of thermotolerance in addition to Hsp70 in vivo as in vitro.