Randomized, double-blind, crossover, placebo-controlled clinical trial to evaluate the effects of chicken hot water extract on insulin secretion.

OBJECTIVE: Essence of chicken (EOC), a hot water extract of chicken, is widely consumed in Southeast Asia as a beverage. EOC has an inhibitory effect on the elevation of blood glucose levels and a secretagogue effect on insulin. However, the mechanism by which EOC promotes insulin secretion is unknown. We aimed to verify the postprandial hyperglycemic inhibitory effect and the insulin secretory effect of EOC in healthy adults under appropriate placebo settings. In addition, we aimed to understand the mechanism underlying the insulin secretory effect of EOC. PATIENTS AND METHODS: Thirty-four healthy Japanese adults were fed 68 mL of EOC or control food, followed by 200 g of cooked rice. Blood glucose and plasma insulin levels were measured at 30, 45, 60, 90, and 120 min after the participants ate cooked rice. The trial had a randomized, double-blind, crossover, placebo-controlled design. RESULTS: The ingestion of EOC induced an increase in the maximum blood concentration (Cmax) of insulin and shortened the time required to reach the maximum blood concentration following rice consumption. Ingestion of the test beverage resulted in a significantly higher insulinogenic index than that obtained after ingestion of the control beverage. No side effects were observed in this study. Mechanistic experiments revealed that EOC stimulated significant (p < 0.05) secretion of GLP-1 from NCI-H716 human intestinal L cells at 0.1, 1, and 10 mg/mL. CONCLUSIONS: Consuming EOC when eating rice supports pancreatic function. Daily consumption of EOC could elevate the early-phase insulin response; therefore, it could prevent diabetes in Asians with low insulin secretion.

Effects of ethyl-esterization, chain-lengths, unsaturation degrees, and hyperthermia on carcinostatic effect of omega-hydroxylated fatty acids.

AIM: To evaluate promotive effect of hyperthermia on the carcinostatic activity of synthesized omega-hydroxy fatty acids (omega HFAs) and their ethylesters agaist Ehrlich ascites tumor (EAT) cells. METHODS: EAT cells were cultured with either omegaHFAs or their ethylester derivatives in a water bath at either 37 degrees C or 42 degrees C for 30 min, followed by incubation in a CO2 incubator for 20 or 72 h. Mitochond-rial dehydrogenase-based WST-1 assay and trypan blue dye exclusion assay were then conducted after incubation. Morphological changes were observed by scanning electron microscopy (SEM). RESULTS: Omega-HFA having a saturated 16-carbon straight-chain (omega H16:0) was the most carcinostatic (at 37 degrees C - viability level: 60.0%; at 42 degrees C - 49.6% (WST-1)) among saturated and unsaturated omegaHFAs with 12, 15 or 16 carbon atoms, when administrated to EAT cells at 100 microM for 20 h. Carcinostatic activity was markedly enhanced by ethyl-esterization of saturated fatty acids, such as omegaH16:0 (at 37 degrees C - 42.3%; at 42 degrees C - 11.2%, ibid) and omegaH15:0 (at 37 degrees C - 74.6%; at 42 degrees C - 25.3%, ibid), and their unsaturated counterparts were extremely effective only in combination with hyperthermia. Prolongation of the incubation period to 72 h at the same concentration increased appreciably their carcinostatic effect (omega H16:0 ethylesther: 1.3%; omegaH15:0 ethylesther: 8.0%). These values were also supported by dye exclusion assay. The carcinostatic activity enhanced more markedly by hyperthermia (1.2%; 2.1%, ibid). SEM shows that omegaH16:0 ethylester-exposed EAT cells underwent extensive injury, such as deformation of cell structure or disappearance of microvilli. CONCLUSIONS: omega H16:0 ethylester possesses high carcinostatic activity in vitro in combination with hyperthermia and may be utilized as potent anticancer therapeutic agent.

Morphological and functional differentiation of human thyroid cells in collagen gel culture.

OBJECTIVE: Cultured human thyroid cells in collagen gel culture were examined on cell morphology and the production of thyroglobulin (Tg), triiodothyronine (T3) and thyroxine (T4) which are components of functional differentiation. METHODS: Thyroid cells obtained from normal human thyroid tissues (four cases), follicular adenoma tissues (three cases), papillary carcinoma tissues (three cases), and follicular carcinoma tissue (one case), were cultured in collagen gel. Then these cultured cells were observed on cellular morphology and production of Tg, T3 and T4. Moreover, changes in morphological characteristics and production of Tg, T3 and T4 induced by addition of thyroid stimulating hormone (TSH) and epidermal growth factor (EGF) to medium in collagen gel culture were determined. RESULTS: Normal and tumor cells in collagen gel culture formed colonies and follicles with Tg production, similar to in vivo-like three-dimensional cellular structures and functions. Normal thyroid cells stimulated TSH induced more Tg and produced morphological changes, i.e. enlarged follicular lumens and increased the height of follicular cells, but did not promote cell proliferation. Reversely, normal thyroid cells stimulated with EGF promoted cell proliferation, but did not change morphological findings and did not increase production of Tg, T3 and T4. CONCLUSIONS: These findings suggest that collagen gel culture is useful for observing the effects of stimulation by cell growth factor on the morphological and functional differentiation of human thyroid cells.

Cathepsin L activity and its inhibitor in human otitis media.

OBJECTIVE: We examined cathepsin L activity, expression of cystatin A, and copper- and zinc-containing superoxide dismutase in human chronic otitis media. The relationships of our findings to clinical findings (e.g., grade of bone destruction) were also studied. DESIGN: Retrospective basic and clinical study. SETTING: Department of Otolaryngology and First Department of Biochemistry, Kinki University School of Medicine, Osaka, Japan. METHOD: The human middle ear tissues evaluated in this study were surgically obtained from seven patients with cholesteatoma epithelium, three patients with granulation tissues in cholesteatoma, three patients with granulation tissues in noncholesteatoma, and three patients with intact mucous membrane of the middle ear. MAIN OUTCOME MEASURES: Cathepsin L activities in cholesteatoma epithelium, granulation tissues in cholesteatoma, or granulation tissues in noncholesteatoma were measured using Barrett's method. Cystatin A expressions were observed by Western blot analysis. Copper- and zinc-containing superoxide dismutase in cholesteatoma was examined immunohistochemically. RESULTS: Mean cathepsin L activity was higher in diseased tissues than in intact mucous membranes of the middle ear. Granulation tissues with high cathepsin L activity resulted in extensive bone destruction in both cholesteatomas and noncholesteatomas of the middle ear. All cases with intact mucous membrane of the middle ear exhibited no expression of cystatin A. Seven of 10 cases with diseased tissues expressed cystatin A in cholesteatoma epithelium, granulation tissues in cholesteatoma, or granulation tissues in noncholesteatoma. No relationships were found between cystatin A expression and grade of cathepsin L activity. Copper- and zinc-containing superoxide dismutase was more strongly positive in cholesteatoma epithelium regions than in granulation tissues. CONCLUSION: These results suggest that copper- and zinc-containing superoxide dismutase in cholesteatoma epithelium prevents complications by suppressing cathepsin L activity.

Type IV collagenase and immunostaining of type IV collagen in human thyroid tumors.

OBJECTIVE: Required stages in the processes of malignant tumor invasion and metastasis are known to include the destruction of cell stroma and vascular basement membrane. It has been suggested that type IV collagenase can degrade type IV collagen, a main component of basement membrane. In our study, type IV collagenase activity was compared with the grade of destruction of type IV collagen by thyroid tumors. METHODS: Type IV collagenase activity was measured in human thyroid tissue obtained surgically from four patients with Graves' disease, four with follicular adenoma, six with papillary carcinoma, and four with follicular carcinoma. Six normal thyroid tissue specimens were also studied. The grade of destruction in the diseased regions of thyroid tumors and surrounding intact tissues were determined immunochemically by anti-type IV collagen antibody staining. RESULTS: Tumors with high type IV collagenase activity exhibited extracapsular invasion, lymph node metastasis and very weak discontinuity of immunostaining for type IV collagen from the basement membrane in diseased regions. In addition, surrounding intact tissues exhibited weak immunostaining for type IV collagen. Tumors with low type IV collagenase activity exhibited neither extracapsular invasion nor lymph node metastasis, but did exhibit weak immunostaining for type IV collagen from the basement membranes in diseased regions. However, the surrounding intact tissues had preserved type IV collagen in follicular basement membranes. CONCLUSION: These findings suggest that staining for type IV collagen can be considered a biochemical marker for prediction of the aggressiveness of invasion and metastasis.

Protective effects of transient HO-1 overexpression on susceptibility to oxygen toxicity in lung cells.

Rat fetal lung cells (RFL-6) were transiently transfected with a full-length rat heme oxygenase (HO)-1 cDNA construct and then exposed to hyperoxia (95% O2-5% CO2) for 48 h. Total HO activity and HO-1 protein were measured as well as cell viability, lactate dehydrogenase (LDH) release, protein oxidation, lipid peroxidation, and total glutathione to measure oxidative injury. HO-1 overexpression resulted in increased total HO activity (2-fold), increased HO-1 protein (1.5-fold), and increased cell proliferation. Immunohistochemistry revealed perinuclear HO-1 localization, followed by migration to the nucleus by day 3. Decreased cell death, protein oxidation, and lipid peroxidation but increased LDH release and glutathione depletion were seen with HO-1 overexpression. Reactive iron content could not explain the apparent loss of cell membrane integrity. With the addition of tin mesoporphyrin, total HO activity was decreased and all changes in injury parameters were normalized to control values. We conclude that moderate overexpression of HO-1 is protective against oxidative injury, but we speculate that there is a beneficial threshold of HO-1 expression.

Immunosuppressive activities of recombinant glycosylation-inhibiting factor mutants.

We have shown previously that glycosylation-inhibiting factor (GIF) in culture supernatants of suppressor T cell (Ts) hybridomas had bioactivity, while the same cells contained a substantial quantity of inactive GIF in cytosol. Mass-spectrometric analysis of GIF in the culture supernatant and cytosol of a Ts hybridoma provided direct evidence that GIF protein was posttranslationally modified in the Ts cells, and that the GIF bioactivity is associated with the posttranslationally modified species. Assuming that conformational changes induced by the posttranslational modifications are responsible for generation of bioactivity, we constructed cysteine mutants of human rGIF (rhGIF) in which cysteine at position 57, 60, or 81 was replaced with Ala, and the mutants were expressed in Escherichia coli. Replacement of Cys57 or Cys60 with Ala resulted in generation of bioactivity, while replacement of Cys81 with Ala failed to do so. It was also found that replacement of Cys57 with Ala and carboxymethylation of a sulfhydryl group in Cys60 synergistically increased the GIF bioactivity of the GIF derivatives. A mutated GIF protein, in which Cys57 and Asn106 in the rhGIF were replaced with Ala and Ser, respectively, had immunosuppressive effects on the IgE and IgG1 Ab responses of BDF1 mice to DNP-OVA, while wild-type rhGIF did not. Evidence was obtained that the mutated GIF suppressed Ag priming of Th cells for the Ab responses and proliferative response.

Malignancies of human thyroid tumors and dynamic magnetic resonance imaging (MRI).

Time intensity curves for gadolinium-diethylene triaminepentacetic acid (Gd-DTPA) enhanced magnetic resonance imaging (MRI), namely dynamic MRI, were determined for thyroid diseases and compared with findings of histopathologic examination. Time intensity curves for solid lesions were determined, excluding cases with secondary changes such as calcification, hemorrhage, necrosis and fibrosis. Three different patterns of time intensity curves were observed: rapid washout, delayed washout and no change. In our previous study, malignant grades of thyroid tumors were estimated immunohistochemically by epidermal growth factor receptor (EGFR) antibody. In most of malignant diseases and a few benign diseases that had marked cell proliferative activity with staining EGFR strongly, the time intensity curve displayed a delayed washout pattern, in which intensity was above 1/2-maximal value within 10 min after injection Gd-DTPA. Almost all benign diseases and a few well differentiated carcinomas displayed a rapid washout pastern, in which intensity was decreased to lower than 1/2 of peak grade within 10 min following injection and showed staining EGFR weakly. Benign diseases showing no change of time intensity curve, did not almost show aEGFR positive cell. These findings suggested that the time intensity curve obtained from dynamic MRI might indicate differentiated grades and cell proliferating activity of thyroid tumors.

High-affinity binding of bioactive glycosylation-inhibiting factor to antigen-primed T cells and natural killer cells.

High-affinity binding was demonstrated between suppressor-T-cell-derived bioactive glycosylation-inhibiting factor (GIF) and helper T hybridomas and natural killer cell line cells. Inactive GIF present in cytosol of suppressor T cells and Escherichia coli-derived recombinant human GIF (rhGIF) failed to bind to these cells. However, affinity of rhGIF for the target cells was generated by replacement of Cys-57 in the sequence with Ala or of Asn-106 with Ser or binding of 5-thio-2-nitrobenzoic acid to Cys-60 in the molecule. Such mutations and the chemical modification of rhGIF synergistically increased the affinity of GIF molecules for the target cells. The results indicated that receptors on the target cells recognize conformational structures of bioactive GIF. Equilibrium dissociation constant (Kd) of the specific binding between bioactive rGIF derivatives and high-affinity receptors was 10-100 pM. Receptors for bioactive GIF derivatives were detected on Th1 and Th2 T helper clones and natural killer NK1.1(+) cells in normal spleen but not on naive T or B cells. Neither the inactive rGIF nor bioactive rGIF derivatives bound to macrophage and monocyte lines or induced macrophages for tumor necrosis factor alpha production.

Novel animal model of lymph node metastasis by intrauterine inoculation of the actively metastatic subline PL3 separated from rat Walker 256 tumor cells.

To investigate the cellular mechanism of lymph node metastasis by tumor cells through the lymphatic vessels in the uterine corpus, we selected an active metastatic subline (PL3) from rat Walker 256 tumor cells and used it to develop a novel experimental model of lymph node metastasis induced by intrauterine inoculation of the tumor cells. Light- and electron-microscopic examinations revealed that the inoculated PL3 cells could actively infiltrate the endometrium from the uterine cavity and form a primary lesion in the uterine corpus. A few PL3 cells in the myometrium were found in the lumen of the peripheral lymphatic vessels on day 7 after inoculation. The regional lymph nodes around the uterus were then invaded by the migrated PL3 cells, and finally (after 3 weeks), most of the parenchyma of the nodes was replaced by metastasized tumor cells. By flow-cytometric analysis, the metastatic PL3 cells expressed CD44, like Walker 256 cells, but lacked integrin alphaL- and alpha4-chains. However, expression of ICAM-1 was considerably down-regulated in the PL3 cells compared to the parent cells. More aggressive invasion was shown by the PL3 cells compared to the parent cells in the in vitro invasion assay. These findings suggest that this experimental model and the separated PL3 cells are suitable for thorough investigations of the unidentified metastatic process and the related cellular behavior involved in the onset of lymphatic invasion by the primary lesion. Furthermore, our model more closely reproduces the clinical conditions related to lymph node metastasis of malignant carcinomas through the lymphatic vessels than does any previously reported animal model.

The crystal structure of human glycosylation-inhibiting factor is a trimeric barrel with three 6-stranded beta-sheets.

Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis. The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method. The structure was refined to an R factor of 0.168 at 1.9 angstrom resolution. The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded beta-sheets on the inside and six alpha-helices on the outside. There is a 5-angstrom-diameter "hole" through the middle of the barrel. The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor. Each subunit has a new class of alpha + beta sandwich structure consisting of two beta-alpha-beta motifs. These beta-alpha-beta motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different.

[Study of type I and IV collagenase activity in human thyroid diseases].

The activity of type I and IV collagenase was measured in thyroid tissue obtained from 6 non-diseased thyroids, 4 patients with Graves' diseases, 5 with follicular adenoma, 6 with papillary carcinoma and 4 with follicular carcinomas. The relationship between these enzyme activities and invasion or metastasis of the original tumors was studied. The activity of type I collagenase in papillary carcinomas and follicular carcinomas was higher than in non-diseased thyroids, Graves' disease and follicular adenoma. Carcinoma tissue with invasion beyond the capsule in particular had higher type I collagenase activity. Type IV collagenase activity in carcinoma with lymph node metastasis was higher than in non-diseased thyroids, Graves' disease and follicular adenoma, and especially higher than carcinoma without lymph node metastasis. These findings suggest that increased type I collagenase activity plays an important role in local invasion in thyroid carcinoma, and that increased type IV collagenase activity plays an important role in lymph node metastasis.

Study on cathepsin B activity in human thyroid tumors.

Cathepsin B activity was measured in human thyroid tissue obtained surgically from 2 patients with Grave's disease, 3 with follicular adenoma, 4 with papillary carcinoma, and 4 with follicular carcinoma. Three normal thyroid tissues were also studied. Comparisons were made between cathepsin B activity and clinical findings, including histopathological diagnosis and the presence or absence of extra-capsular invasion and metastasis. The abilities of original tumors to degrade type I and type IV collagen were also measured. Mean cathepsin B activities of both specimens with benign and those with malignant disease were significantly higher than those of normal thyroid. On cases of thyroid carcinoma, those with extra-capsular invasions and metastasis had the highest cathepsin B activities. Cases with high cathepsin B activities also tended to show high type I and IV collagen degrading abilities. These findings suggest that cathepsin B plays a role in the development of extra-capsular invasion and lymph node metastasis in human thyroid tumors.

Productivity of thyroglobulin and thyroid hormone on human thyroid cell in collagen gel culture.

Thyroid cells obtained from 21 normal human thyroid tissue samples and 17 tissue samples from diseased thyroid, including one of Graves disease, 4 with follicular adenoma, 11 with papillary carcinoma, and 1 with follicular carcinoma, were cultured in collagen gel, and ability to produce thyroglobulin (Tg), triiodothyronine (T3) and thyroxine (T4) was determined. Changes in morphological characteristics and production of Tg, T3, and T4 induced by addition of thyroid stimulating hormone (TSH) to medium in collagen gel culture were also determined. Twenty of all cases exhibited positive reaction for Tg. No relationship was found between rate of positivity for Tg and pathologic diagnosis. Three with carcinoma showed positive reaction for T3, and 4 with carcinoma showed positive reaction for T4. Only for normal thyroid cells did addition of TSH to medium induce increase the percentage of colonies producing Tg or T4 and morphological changes including an enlarged follicular lumen and increase in the height of columnar epithelium. These findings suggest that thyroid cells in collagen culture develop in an in vivo-like fashion. In conclusion, collagen has important effects on cellular differentiation when included in extracellular matrix.

[Histopathological findings of human thyroid tumors and signal intensities of magnetic resonance imaging (MRI)].

We studied the correlation between postoperative thyroid histological findings and signal intensities of preoperative magnetic resonance (MR) imaging. The study subjects included 20 cases with thyroid diseases (7 with adenomatous goiter, 2 with follicular adenoma, 6 with papillary carcinoma, 5 with follicular carcinoma). Solid diseases excluding secondary changes (calcification, hemorrhage, necrosis and fibrosis) were investigated by T1 weighted imaging (T1WI) and T2 weighted imaging (T2WI). The results of this study showed that high signal intensities on T1 and T2 indicated preserved follicular structures with a full colloid and sparse atypical cells, in both benign and malignant diseases. Trabecular and microfollicular structures that showed destruction of follicular components and sparse colloid, had a tendency to show low approximately iso signal intensities on T1WI imaging. These results suggest that differentiated grades and the cell proliferative ability of thyroid tumors may be predictable with a combination of T1WI and T2WI.

[Proliferating cell nuclear antigen (PCNA) on human diseased thyroid cells].

Surgical materials obtained from 5 normal human thyroids and 19 diseased tissues (4 with Grave's disease, 4 adenomatous goiters, 3 follicular adenomas, 2 papillary carcinomas, 4 follicular carcinomas and 2 undifferentiated carcinomas) were investigated immunohistochemically using the avidin-biotin-peroxidase complex technique with anti-proliferating cell nuclear antigen (PCNA) antibody and epidermal growth factor receptor (EGFR) antibody. The results of this study suggested the following: (1) Labeling indexes of PCNA positive cell nuclei were shown in high grade malignant cases accompanied by capsular invasion and lymph node metastasis, in undifferentiated carcinoma cells, and in proliferative lesions of benign diseases. These results suggest that PCNA is indicative of proliferative ability in thyroid diseases. (2) On thyroid tumors, EGFR regions with strong positive staining showed many PCNA positive cell nuclei. EGF appears to have a significant association with proliferative ability in thyroid diseases.

Sporulation-inhibitory gene in pock-forming plasmid pSA1.1 of Streptomyces azureus.

A plasmid, pSA1.1, of Streptomyces azureus elicited conjugative pocks and inhibited the sporulation of its host mycelia. pSA1.1 was studied with a kanamycin resistance gene derived from S. kanamyceticus as a selective marker. The deletion analysis and sequencing of the derivative plasmid found an open reading frame (ORF909b) that was involved in the sporulation-inhibitory function of pSA1.1 and in pock formation. The nucleotide sequences of ORF909b were compared with those of the genes registered in GenBank, but no similarity was found. However, the predicted amino acid sequence of ORF909b showed a significantly high similarity with that of the spoIIIE gene of Bacillus subtilis. This detected gene might be a new sporulation-regulatory gene in streptomycetes.

[Study on human diseased thyroid cells in collagen gel culture--morphology and their malignancy of cultured colonies].

Thyroid cells from 14 normal subjects, two patients with Grave's disease, four patients with follicular adenoma and eight patients with papillary carcinoma were cultured in collagen gel. The colonies of these cells were stereoscopically observed and their morphological characteristics were studied with regard to relation with pathological findings of mother tumor, extra-capsular invasion and metastatic potential. For normal thyroid, Grave's disease and follicular adenoma (except for one case), their own characteristic branching type colonies were found. For papillary carcinoma, both branching type and spheroid type of colonies were observed. The ratio of branching type/spheroid type varied individually in the patients with papillary carcinoma. However, the spheroid type was found to trend to be predominant in patients with extra-capsular invasion and/or lymph node metastasis. This means that the observation of spheroid type colonies in collagen gel is suggestive of risk of extra-capsular invasion or lymph node metastasis. From the obtained results, it seemed possible to diagnose poorly-differentiated cells in vitro by morphologically observing colonies of human thyroid papillary carcinoma cells developing in collagen gel.

[Morphological study of human thyroid papillary carcinoma cells in collagen gel culture].

Human thyroid papillary carcinoma cells (surgical materials) were cultured in a monolayer or a collagen gel system. In the monolayer culture, degeneration and necrosis of cancer cells were observed after 10 days of culture. In the collagen gel culture, degenerative necrosis were observed 14 days culture. In histopathological observation monolayer cells proliferated in pavement-like form without forming any colony. On the contrary, collagen gel culture cells formed branching-type colonies and spheroid-type colonies. Those results suggested that papillary carcinoma cells can be cultured more like in vivo in collagen gel culture than monolayer culture.

Endonuclease activity on damaged DNA in rat regenerating liver.

The activity of an endonuclease(s) acting on double-stranded, ultraviolet-irradiated, and 2-acetylaminofluorene-bound DNA but not on double-stranded undamaged DNA triples within two hr after partial hepatectomy. Although the activity drops between four and six hr after operation, it remains above levels measured in livers of nonhepatectomized rats until 36 hr after operation. Between 36 and 48 hr after operation, the enzyme activity drops below the levels in liver of nonhepatectomized rats and then rises slowly to reach levels observed in nonhepatectomized animals between 48 hr and seven days after the operation. Studies on the effect of actinomycin on the activity of crude enzyme and on the incorporation of [14C]leucine and [14C]valine on the purified enzyme indicate that the increase in enzyme activity results from de novo synthesis. Eight % of endonucleolytic activity detectable in the crude homogenate is inhibited by an hyperimmune serum prepared against the purified enzyme. By adjusting the time of injection of 2-[14C]acetylaminofluorene with respect to the levels of enzyme activity after partial hepatectomy, an inverse correlation between binding and enzyme activity was demonstrated.