Anti-cancer and pro-apoptotic effects of an herbal medicine and Saccharomyces cerevisiae product (CKBM) on human hepatocellular carcinoma HepG2 cells in vitro and in vivo.

Hepatocellular carcinoma is a major health problem worldwide. Different treatment strategies have been developed to cope with this problem. Herbal medicine is now widely studied in both Eastern and Western countries. In this study, we used both in vitro and in vivo model to illustrate the anti-tumor effect of a product, CKBM, consisting of herbal medicine and specially processed Saccharomyces cerevisiae. Dose-dependent anti-proliferation effect was observed on in vitro growth of human hepatoma HepG2 cells after 48 hours incubation with CKBM. At the 50% inhibitory concentration (IC50) no significant toxic effect was observed on normal human fibroblasts Hs68 and human liver WRL-68 cells. The results of morphological changes, detection of DNA fragmentation, flow cytometric analysis and Western blot analysis indicated that this anti-tumor effect of CKBM was mediated via the process of apoptosis. In addition, HepG2 cells- bearing nude mice model was used for in vivo anti-tumor study. Our results showed that 14-day treatment with 0.8 ml daily dosage of CKBM could inhibit 54.1% of tumor growth. The plasma activities of enzymes specific for heart and liver, namely creatine kinase, lactate dehydrogenase, aspartate transaminase and alanine transaminase, remained at normal levels, indicated that CKBM did not produce toxicity to the host.

Modulation of the Ca(2+)-activated Cl(-) channel by 14-3-3epsilon.

We have previously reported an association of 14-3-3epsilon isoform with calmodulin. Using the voltage-clamp technique, the present study investigated the potential role of 14-3-3 in modulating the Ca(2+)-activated Cl(-) channel (CaCC) endogenously expressed in Xenopus oocytes. Injection of 14-3-3epsilon antisense oligodeoxynucleotides resulted in potentiation of the ionomycin-induced Cl(-) current, while 14-3-3 peptide and calmodulin inhibitor, W13, suppressed the antisense-potentiated current. The data suggest that 14-3-3epsilon plays an inhibitory role in modulating the CaCC by interacting with the calmodulin-dependent pathway. The potential role of 14-3-3epsilon in other tissues and its therapeutic potential for cystic fibrosis are discussed.

In vivo and in vitro association of 14-3-3 epsilon isoform with calmodulin: implication for signal transduction and cell proliferation.

Using a yeast two-hybrid screen, human 14-3-3 epsilon protein was found to interact with human calmodulin. In vitro binding assay between human 14-3-3 epsilon protein/peptide and calmodulin was demonstrated by native gel electrophoresis, and the interaction was shown to be calcium dependent. Our results, along with the association of the 14-3-3 epsilon protein with other signaling proteins, suggest that the 14-3-3 protein could provide a link between signal transduction and cell proliferation.

Molecular cloning and characterization of FHL2, a novel LIM domain protein preferentially expressed in human heart.

A full-length cDNA clone encoding a novel LIM-only protein was isolated and sequenced from a human fetal heart cDNA library. This full-length clone consists of 1416 base pairs and has a predicted open reading frame (ORF) encoding 279 amino acids. The ORF of this polypeptide codes for the human heart-specific four and a half LIM-only protein 2 (FHL2). It possesses an extra zinc finger that is a half LIM domain and four repeats of LIM domain. When the human FHL2 cDNA probe was used to hybridize with poly-A RNA of various human tissues, a very strong signal could be seen in heart tissues, and only moderately low signals could be detected in placenta, skeletal muscle and ovary. Virtually no signal could be detected in brain, lung, liver, kidney, pancreas, spleen, thymus, prostate, testis, small intestine, colon or peripheral blood leukocyte. FHL2 was mapped to chromosome 2q12-q13 by fluorescent in-situ hybridization (FISH).

Developmental regulation of 14-3-3 epsilon isoform in rat heart.

Human heart cDNA sequencing yielded a cDNA clone that is similar in DNA and amino acid sequences to that of mouse 14-3-3 epsilon isoform. The 6xHis-tagged H1433 epsilon recombinant protein was expressed in Escherichia coli and its size was approximately 30 kDa. From Northern blot results with human multiple tissues, human skeletal muscle was found to have the highest level of h1433 epsilon mRNA expression, whereas Northern blots of human cancer cell lines detected the highest mRNA level of h1433 epsilon in colorectal adenocarcinoma SW480. The protein expression level of h1433 epsilon and Raf-1 is found to be regulated coordinately during rat heart development, and their protein expression was highest from 14.5 to 16.5 days postcoitum.

Isolation and characterization of a human heart cDNA encoding a new member of the small heat shock protein family--HSPL27.

A novel cDNA clone was isolated from a human adult heart cDNA library. This cDNA clone is similar to the small heat shock protein (smhsp) in both DNA and amino acid sequences, especially in the conserved region. Sequence analysis has shown that the putative novel smhsp, named 27 kDa heat-shock-protein-like protein (HSPL27) is a protein of 241 amino acids with a deduced molecular mass of 26.7 kDa and a deduced pI of 8.0. We have expressed the HSPL27 in E. coli and the expressed protein was found to be present in the soluble fraction of the bacterial cell lysate. Chromosomal mapping data shows that the HSPL27 gene is located at human chromosome 5q11.2.

A novel cDNA encoding a human homologue of ribosomal protein L29.

During the large scale partial sequencing of human heart cDNA clones, a novel clone which is very similar to the rat ribosomal protein L29 in both DNA and amino acid sequences was found. The cDNA encodes a protein with a deduced molecular weight of 17751 (159 aa). It shows 80.4% homology to protein L29 from the large ribosomal subunit of rat and is related to yeast YL43. The putative protein was named human ribosomal protein L29 (hRPL29). hRPL29 has a large excess of basic residues over acidic ones. The large amount of charged residues makes the protein very hydrophilic and the protein has a deduced pI of 12.16. Internal repeats have been characterised in many ribosomal proteins and a tandem repeat of KAKAKAKA was found to be unique to hRPL29. Analysis of gene organisation by Southern blotting shows that of the approximate 10 copies of hrpL29, all but one are pseudogenes. Northern analysis indicated that the mRNA that encodes human L29 is approx. 800 base pairs in length. An intron of hrpL29 has also been cloned and sequenced by polymerase chain reaction using human genomic DNA as the template.

The importance of extensive sampling and examination of cervix in suspected cases of amniotic fluid embolism.

Amniotic fluid embolism is an important complication of pregnancy with high mortality. The diagnosis of amniotic fluid embolism is generally made postmortem and rests upon the histological demonstration of amniotic fluid debris, including foetal epithelial squames and hair, in the pulmonary vasculature. We have made the diagnosis of amniotic fluid embolism in two patients by detection of the amniotic fluid debris in the blood vessels of the cervix in their hysterectomy specimens. These two patients presented with profuse primary postpartum haemorrhage and evidence of disseminated intravascular coagulation after uneventful deliveries. Amniotic fluid debris were only demonstrated in the blood vessels of the cervix but not in the corpus. This observation emphasizes the importance of a thorough histological examination of the cervix in cases of suspected amniotic fluid embolism.

Lipoma and liposarcoma of the parotid gland: high-resolution preoperative imaging diagnosis.

Over the past 7 years, nine fatty tumors within the parotid gland have been managed (eight lipomas, one liposarcoma). High-resolution computed tomography examination was carried out in all cases; with correct preoperative diagnosis recorded each time. The computed tomography imaging characteristics of lipoma, liposarcoma, and the differential diagnosis from other fat density lesions, such as a fatty infiltration, appear quite specific. The liposarcoma and six of the lipomas were resected at formal parotidectomy with facial nerve preservation. Two patients with small intraglandular lipomas have elected to undergo long-term clinical and imaging observation. Our experience indicates that high-resolution, soft-tissue imaging with computed tomography and magnetic resonance imaging permits consistent preoperative fatty tumor diagnosis. This imaging input facilitates rational treatment decision-making.

Complement-mediated pulmonary edema in sheep.

Incubation of plasma with zymosan results in complement activation. Infusion of zymosan-activated plasma (ZAP) into the pulmonary circulation in sheep results in a transient rise in pulmonary vascular resistance, accompanied by hypoxemia. This is associated with production of the vasoactive prostaglandin metabolite, thromboxane. We hypothesized that ZAP infusion caused pulmonary edema and may transiently alter pulmonary permeability to protein. Two sets of experiments were conducted involving six sheep. Three sheep underwent open lung biopsies during ZAP infusion, and electron microscopy documented early reversible interstitial pulmonary edema in response to ZAP infusion. Three other sheep were prepared with lung lymph fistulae and subjected to a similar infusion of ZAP. These studies documented a rapid rise in lymph flow and lymph protein clearance, occurring immediately upon infusion of ZAP. These experiments demonstrate a rapid reversible interstitial edema in sheep lung in response to ZAP infusion and suggest that permeability to protein may be transiently altered as a result of this injury.

Aggressive thyroid cancer in low-risk age population.

Seventy-eight patients under the age of 40 (low-risk patients) who had undergone surgical treatment for well-differentiated thyroid carcinoma were referred from 1979 to 1986 to our hospital for adjuvant therapy. A subgroup of 37 patients, 14 with apparent aggressive cancer, was studied. This study group consisted of 27 female and 10 male patients with mixed papillary and follicular cancer, who ranged in age from 11 to 40 years. Nodal disease occurred in 27 (73%) patients and invasiveness in 30 (81%) patients and involved multiple areas in 9 (24%) patients. Recurrence occurred in 14 (38%) patients and visceral metastases occurred in eight (22%) patients. All patients underwent appropriate surgery, but microscopic residual disease was seen in 15 patients and gross residual disease in seven patients, so that 31 patients underwent iodine-131 therapy, and 17 of these patients also underwent external radiation therapy. Three patients died of their disease, whereas 24 (65%) patients are free of disease and 9 (24%) patients are alive with disease. An additional 7 (19%) patients were initially seen in the fifth to seventh decade after decades of neglected thyroid disease, which culminated in residual cancer and death. Although low-risk categorization for thyroid cancer appears valid, its rigid application in support of conservative treatment may lead to inadequate primary treatment and underdiagnosis of cancer in thyroid nodule disease in the low-risk age population.

Metamorphosis of dermal cylindroma: possible relation to malignant transformation. Case report of cutaneous cylindroma with direct intracranial invasion.

A case of locally invasive cutaneous cylindroma is reported. This neglected scalp tumor, 6.5 cm in greatest dimension, initially invaded the calvaria at the time of the first operation. A recurrence infiltrated the dura and presented as an intracranial space-occupying lesion. No brain invasion was present and distant metastases were not documented. The patient remains well 2 1/2 years after tumor resection. Light and electron microscopic appearance of the recurrent tumor revealed distinct morphologic features, which may be of use in predicting the biologic behavior of cutaneous cylindromas. This case documents the low biologic aggressiveness of cylindroma and stresses the necessity of complete surgical excision of neoplasms with atypical morphologic features.

Induction of hyperparathyroidism by radioactive iodine.

External irradiation to the head and neck has been associated with the emergence of hyperparathyroidism, due both to adenoma and hyperplasia since a single case report was published in 1975. A recent report indicates that there may be some association between hyperparathyroidism and radioiodine administered for thyroid disease. Eight patients are presented herein, all female and in middle age, who presented with hyperparathyroidism due to adenoma (six patients) or hyperplasia (one patient who had undergone radioiodine treatment). In three patients, the dose of radioiodine exceeded 30 mCi. The duration of latency ranged from 4 to 20 years. Associated thyroid disease included cancer, colloid nodules, and thyroiditis. All patients became eucalcemic and have remained so. Experimental results that have supported the influence of radioiodine on parathyroid hyperactivity do not appear completely applicable to the human model. It is believed that the low mitotic rate equivalent to low radiosensitivity may have protected the parathyroid gland somewhat from neoplastic change, although the association of thyroid carcinoma and parathyroid adenoma appears to be a significant expression of radiation exposure. It may be the lack of a pituitary-stimulating hormone and the absence of an abscopal mechanism, as illustrated by irradiation to the thyroid, that explains the relative immunity of parathyroid behavior in the face of radioiodine exposure. Regardless of the frequency and validity of the association, it is important to recognize the possibility of the association illustrated by our case reports and scrutinize appropriate patients by intermittent serum calcium sampling, both for confirmation and possible detection of occult hyperparathyroidism.

Lymphoma. Presenting as an intramuscular small cell malignant tumor.

The authors report two patients who presented with a mass in their thigh muscles. On the basis of the histologic appearance, a diagnosis of small cell malignancy was made. Further investigations including electron microscopy and immunoperoxidase techniques were necessary to accurately identify these tumors as lymphoma. Intramuscular lymphoma is rare, but it should be considered in the differential diagnosis, when the tumor consists of small cells. Special techniques are mandatory for a definitive diagnosis.

Effects of methylprednisolone and azathioprine on bronchial healing following lung autotransplantation.

The effect of low-dose immunosuppressive therapy upon the healing of the bronchial anastomosis and skin wound following lung autotransplantation was evaluated. Autotransplantation was performed in two groups of dogs: Group 1 (15 dogs) received no immunosuppression and Group 2 (13 dogs) received postoperative immunosuppression with methylprednisolone (2 mg/kg) and azathioprine (1.5 mg/kg). Two to four skin incisions 7 cm in length were made in the dorsal region of each dog. Dogs were put to death at 4, 9, 16, and 23 days postoperatively and the bronchial anastomoses and skin wounds were evaluated by breaking strength measurements. Bronchus and skin breaking strength increased with time in both groups. Bronchus breaking strength was similar in the two groups at day 4 and day 9. However, by day 23 bronchus breaking strength was significantly higher in Group 1 (p less than 0.001). Skin breaking strength was significantly higher in Group 1 on days 9 and 16 (p less than 0.005) and on day 23 (p less than 0.001). Our results suggest that low-dose methylprednisolone and azathioprine significantly affect the breaking strength of both bronchial anastomoses and healing skin incisions following canine lung autotransplantation.