Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection.

Successful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1-4) did not increase, even at 180 min (0.7-4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120-180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0-60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39. The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.

Antibacterial activity of polyphenol components in oolong tea extract against Streptococcus mutans.

The purpose of the present study was to determine the antibacterial activity of oolong tea extract on oral streptococci, including Streptococcus mutans and Streptococcus sobrinus, and to identify the response to its components. Antibacterial activity was found when the extract was added to S. mutans cells in chemically defined medium but not in complex broth media. Further, pretreatment with bovine serum albumin reduced the antibacterial activity. The extract showed antibacterial activity against all of the oral streptococci examined, with the highest activity against S. mutans MT8148R. This activity was found to originate from a monomeric polyphenol-rich fraction, and it was stronger than that of pure polyphenols. Moreover, some combinations of monomeric polyphenols showed the highest level of antibacterial activity. These results suggest that the antibacterial activity of oolong tea extract is caused by a synergistic effect of monomeric polyphenols, which can easily bind to proteins.

Hepatocellular carcinoma: involvement of the internal mammary artery.

PURPOSE: To investigate factors related to the development of internal mammary arteries (IMAs) as feeding arteries of hepatocellular carcinomas (HCCs). MATERIALS AND METHODS: In 30 patients with HCC located in ventral hepatic areas directly beneath the diaphragm, bilateral internal mammary arteriography was performed to explore involvement of the IMA with HCC. The number of previous transcatheter arterial embolizations (TAEs), tumor size, time from initial TAE to IMA angiography, inferior phrenic artery (IPA) involvement with tumor, presence of hepatic artery occlusion, and use of other treatments were compared in groups with and without involvement of the IMA. RESULTS: The group with IMA involvement included 10 patients; the group without involvement, 20 patients. TAE had been performed two to 12 times in the group with involvement and zero to six times in the group without involvement (P =.01). Mean tumor sizes in these two groups were 5.1 and 6.0 cm, respectively; hepatic artery occlusion was noted in nine and zero patients (P =.01) in the two groups. The time from initial TAE to IMA angiography ranged from 3 to 53 months (median, 31.5 months) and from zero to 89 months (median, 0 months) (P =.01). IPA involvement was observed in seven and four patients (P =.015). CONCLUSION: These results strongly suggest that, regardless of tumor size, when HCCs are located in the ventral hepatic areas directly beneath the diaphragm, the IMAs serve as feeding arteries in patients with hepatic artery occlusion caused by repeated TAE.

Prevalence of large-joint osteoarthritis in Asian and Caucasian skeletal populations.

OBJECTIVE: To determine ethnic variations of large-joint osteoarthritis (OA) in past populations. METHODS: One thousand two hundred and nine adult skeletons, excavated from archaeological sites in Japan, China and France were assessed for OA as defined by the presence of eburnation. RESULTS: Within Asian skeletal populations, elbow OA and patellofemoral joint OA were more common in hunter-gatherers than in agriculturalists. Compared with Caucasians, the Asian skeletal population had a higher prevalence of tibiofemoral joint OA. CONCLUSION: The relative frequencies of OA within and between ethnic groups at certain joint sites have changed over time from the past to the present.

Did ossification of the posterior longitudinal ligament of the spine evolve in the modern period? A paleopathologic study of ancient human skeletons in Japan.

OBJECTIVE: To investigate the historical origins of ligamentous ossifications of the spine in Japan. METHODS: We studied skeletons of Jomonese of the period 5000-2300 years BP, of Japanese of the 17th-19th century Edo Period, and of Ainu of the 18th-20th century in Japan with special emphasis on spinal ligament ossifications as in the posterior longitudinal ligament. A comparison to our previous study on ancient Chinese skeletons was done. RESULTS: Cervical ossification of the posterior longitudinal ligament (OPLL) was the only ossification that increased significantly in prevalence in people of the near-modern period in comparison to the Neolithic gathering-hunting people. CONCLUSION: Socioeconomic changes from a subsistence gathering-hunting economy to the near-modern livelihood depending on rice-eating and a diet high in vegetable protein are speculated to be responsible for the prevalence increase of cervical OPLL.

Calcium Dependence of Settlement and Nematocyst Discharge in Actinulae of the Hydroid Tubularia mesembryanthemum.

The influence of Ca2+ and Mg2+ ions on both atrichous isorhiza (AI) discharge and settlement of actinular larvae of the hydroid Tubularia mesembryanthemum was investigated. Mg2+-supplemented artificial seawater (ASW) completely inhibited both events at a concentration of 206 mM, whereas lowered Mg2+ concentrations enhanced them. Ca2+ ions in the bathing solution highly regulated AI discharge and settlement, and Mg2+ ions may down-regulate these events. The effect of inorganic Ca2+-channel blockers, including Gd3+ and La3+, was also examined. Larval settlement was inhibited by Co2+, Ni2+, Cd2+, La3+, and Gd3+, with half inhibitory concentrations (IC50) of 5800, 260, 53, 45, and 7 {mu}M, respectively; AI discharge was also inhibited by these ions, with IC50 values of 6600, 500, 78, 41, and 5 {mu}M, respectively. These results suggest possible involvement of stretch-activated Ca2+ channels in the signal transmission of both AI discharge and larval settlement.

Restoration of holoceruloplasmin synthesis in LEC rat after infusion of recombinant adenovirus bearing WND cDNA.

Wilson's disease, an autosomal recessive disorder, is characterized by the excessive accumulation of copper in the liver. WND (ATP7B) gene, which encodes a putative copper transporting P-type ATPase, is defective in the patients. To investigate the in vivo function of WND protein as well as its intracellular localization, WND cDNA was introduced to the Long-Evans Cinnamon rat, known as a rodent model for Wilson's disease, by recombinant adenovirus-mediated gene delivery. An immunofluorescent study and a subcellular fractionation study revealed the transgene expression in liver and its localization to the Golgi apparatus. Moreover, since the synthesis of holoceruloplasmin is disturbed in the Long-Evans Cinnamon rat, the plasma level of holoceruloplasmin, oxidase-active and copper-bound form, was examined to evaluate the function of WND protein with respect to the copper transport. Consequently, the appearance of holoceruloplasmin in plasma was confirmed by Western blot analysis and plasma measurements for the oxidase activity and the copper content. These findings indicate that introduced WND protein may function in the copper transport coupled with the synthesis of ceruloplasmin and that the Golgi apparatus is the likely site for WND protein to manifest its function.

Intrathoracic hyperthermochemotherapeutic perfusion for the intrathoracic malignancies in gastric cancer.

BACKGROUND/AIMS: We introduced intrathoracic hyperthermochemotherapeutic perfusion to two patients with intrathoracic lesions in gastric cancer, one patient with pleural dissemination and the other with left lung metastases. MATERIALS AND METHODS: Heated saline containing cisplatin, mitomycin C, and etoposide was circulated through the patient's thorax by a magnetic pump for an hour. RESULTS: The effects of this procedure were estimated complete respose in the former and no change in the latter. There were no complications. While the intrathoracic temperatures were stable between 42.0 and 42.5 degrees C, systemic temperatures measured at esophagus, urinary bladder, pulmonary artery, and tympanic membrane very gradually increased and peaked under 38.6 degrees C. The drug concentration in the perfusate was much lower than the peak plasma concentration, though concentration in the plasma was higher than the peak plasma concentration. CONCLUSIONS: This procedure is one of the preferable methods for treatment of intrathoracic lesions in gastric cancer because of easy temperature control, pharmacological advantage, and lack of side effects.

[Chemohyperthermic peritoneal perfusion and subtotal peritonectomy for peritonitis carcinomatosa in gastroenteric cancer].

A new operative procedure, called subtotal peritonectomy (SP), in combination with chemohyperthermic peritoneal perfusion, was developed for the treatment of peritonitis carcinomatosa in gastrointestinal cancer. SP includes resection of primary lesion, colon, small bowel, spleen, and gall bladder and parietal peritonectomy. Six patients with gastric cancer and two patients with colon cancer underwent these procedures. A great deal of discharge from the peritoneal cavity, an increase in systemic vascular resistance index, and a decrease in central venous pressure represented much decrease in circulatory volume on days 1 to 2 postoperatively. This state improved at 3 to 4 days after operation. Histopathological study revealed multiple peritoneal seedings with negative surgical margins in all patients. There were no related deaths though bleeding, perforation, and abscess occurred in two patients each. One patient died of peritoneal recurrence after one year, but the other have survived.

Isolation and characterization of the cDNA for pea chloroplast SecA. Evolutionary conservation of the bacterial-type SecA-dependent protein transport within chloroplasts.

We report here the isolation of the cDNA for pea chloroplast SecA. Pea SecA encodes a polypeptide of 1,011 amino acids and shows high sequence similarity with cyanobacterial SecA. Pea SecA was synthesized as a larger precursor and was imported into isolated chloroplasts in vitro. The purified pea SecA, which was expressed in Escherichia coli cells, stimulated the in vitro import of the 33 kDa protein of the oxygen-evolving complex into thylakoids. These results indicate that higher plant chloroplasts contain a bacterial-type SecA protein-dependent system for the intraorganellar protein transport into thylakoids.

Antiarrhythmic effects of eicosapentaenoic acid during myocardial infarction--enhanced cardiac microsomal (Ca(2+)-Mg2+)-ATPase activity.

The effects of dietary supplementation with eicosapentaenoic acid (EPA) on ventricular arrhythmias during myocardial infarction were examined in a canine model. EPA was incorporated into cellular membranes after ingestion of EPA-ester (100 mg/kg body weight/day) for 8 weeks. The ratio of EPA to arachidonic acid (AA) in platelet cell membranes and myocardial microsomes was significantly increased (7% to 37% in platelet cell membranes; p < 0.01, 3% to 12% in non-infarcted cardiac microsomes; p < 0.01, and from 2% to 8% in infarcted cardiac microsomes; p < 0.01). Dietary supplementation with EPA significantly reduced the incidence and severity of arrhythmias during coronary artery occlusion. Immediately after coronary artery occlusion, all of the animals in the control group that were given a toxic dose of digitalis developed ventricular tachycardia (VT) or ventricular fibrillation (Vf), whereas none of the animals in the EPA-supplement group developed VT or Vf within 15 min after administration of digitalis. Regardless of the presence of an infarcted area, the specific activity of the Ca(2+)-pump enzyme ((Ca(2+)-Mg2+)-ATPase) within the myocardial microsomal fraction of the EPA-supplemented group was significantly higher than in that of the control group (Vmax: 140.5 +/- 19.1 vs 94.8 +/- 28.9 nmol/mg/min in non-infarcted cardiac microsomes, p < 0.01, 130.9 +/- 18.4 vs 90.2 +/- 26.4 nmol/mg/min in infarcted cardiac microsomes, p < 0.01, EPA vs control group, respectively). The specific activities of the Na(+)-pump enzyme ((Na(+)-K+)-ATPase) and NADPH-dependent cytochrome C reductase in infarcted and non-infarcted cardiac microsomes did not differ between these groups. These results indicate that EPA supplementation increases the (Ca(2+)-Mg2+)-ATPase activity within myocardial membranes that is involved in Ca2+ metabolism in myocardial cells by increasing the ratio of EPA to AA within cellular membranes. These cellular alterations are likely to reduce the severity of ventricular arrhythmias by inhibiting the rapid accumulation of intracellular Ca2+ following ischemia.

Chloroplast protein import. Chloroplast envelopes and thylakoids have different abilities to unfold proteins.

Proteins have to be at least partially unfolded upon passage through the biological membranes. Previous studies with a dihydrofolate reductase fusion protein containing a chloroplast transit peptide showed that stabilization of the tertiary structure of the fusion protein by binding of a ligand, methotrexate, failed to block its translocation across the envelopes, suggesting that chloroplast envelopes have strong activity to unfold proteins [America, T., Hageman, J., Guéra, A., Rook, F., Archer, K., Keegstra, K. & Weisbeek, P. (1994) Plant Mol. Biol. 24, 283-294]. In the present study, we have analyzed in vitro translocation of a fusion protein consisting of the entire plastocyanin precursor and dihydrofolate reductase across the chloroplast envelope membranes and the thylakoid membrane. In the presence of methotrexate, the fusion protein was imported into the stroma but its translocation across the thylakoid membrane was blocked. The fusion protein that bound to the envelope became susceptible to digestion by thermolysin. These results suggest that, while the envelope membranes can unfold the methotrexate-bound fusion protein to allow its passage, the thylakoid membrane cannot unfold the fusion protein that has re-bound to methotrexate in the stroma.

[Dual arterial infusion chemotherapy, continuous hyperthermic peritoneal perfusion, and total parietal peritonectomy for peritoneal dissemination in gastric cancer--a case report].

A 36-year-old man developed massive ascites, of which cytology proved Class V, and upper gastrointestinal endoscopy disclosed Borrmann type 4 gastric cancer. We performed dual arterial infusion chemotherapy (DAIC) through the celiac axis and superior mesenteric artery, followed by the operation including continuous hyperthermic peritoneal perfusion, total gastrectomy, subtotal colectomy, massive intestinal resection, splenectomy, cholecystectomy, appendectomy, and total parietal peritonectomy (TPP). Ascites disappeared and the CA125 level was normalized by DAIC. This operation was well tolerated. Though the histopathological study revealed multiple peritoneal disseminations on the visceral peritoneum of stomach, small intestine, colon, and parietal peritoneum such as Douglas' pouch, there were denatured cancer cells in the visceral peritoneum to which blood supply under DAIC was distributed. These results suggested the effect of neoadjuvant DAIC and the significance of TPP.

Pathological studies on local tissue reactions in guinea pigs and rats caused by four different adjuvants.

We investigated pathological changes at the injection site in guinea pigs and rats for 16 weeks following a single intramuscular injection of one of the following oil adjuvant emulsions; oil adjuvant ISA-70, Freund's incomplete adjuvant, Freund's complete adjuvant, and aluminium phosphate gel. In the animals injected with ISA-70 emulsion prepared by manual shaking, grossly, there was partial thickening of subcutaneous tissue, discoloration of inter-muscular connective tissue, and swelling of the inguinal lymph nodes at 2 and 4 weeks post injection (PI). Histopathologically, ISA-70 injected sites revealed acute inflammatory changes at 72 hrs PI, and peak reactions consisting of macrophage accumulation around oil cysts and fibrosis were observed at 4 weeks PI. These changes were less severe and of shorter duration than those in the other three adjuvants. Guinea pigs and rats injected with materials containing inactivated Newcastle disease virus (NDV) antigen similarly showed an infiltration of plasma cells and lymphocytes in addition to the changes described above. ISA-70 containing NDV antigen induced similar hemagglutination-inhibition titer to that induced by Freund's incomplete adjuvant.

[Three adult intussusception cases with colorectal cancer; CT diagnosis and its characteristic findings].

We have experienced three adult intussusception cases with colorectal cancer. In all three cases CT studies were performed prior to administering a barium enema, where we could make the exact diagnosis of the lesions in both the plain and the contrast studies. In the CT studies, the intussusception lesions showed the characteristic eccentric layered pattern. It was difficult to distinguish the tumors from the edematous intestinal walls at the proceeding portion in two cases. While in one case, the proceeding portion (the tumor itself) was pointed out as the enhanced soft tissue density mass of layered pattern with the irregular margins.

[Effects and indications of spinal cord stimulation on the vegetative syndrome].

The effects of spinal cord stimulation (SCS) on the vegetative syndrome were studied in six patients. Factors affecting the results were mentioned with a view to establishing indications as to whether or not the SCS should be performed. "Persistent vegetative states" were thought to be identical with Ohta's "vegetative syndrome" which consists of eleven signs. Six of these signs--polyphasic cycle of waking and sleeping, urinary incontinence, being bedridden and being tube fed etc--were important criteria of the vegetative syndrome. SCS was thought to be effective if one or more of the 6 signs disappeared after SCS. SCS was performed at level from C2 to C4 with a frequency of 25 to 120 Hz, an intensity of 2.5 to 6 volts, a pulse duration of 0.3 to 0.5 msec. and a duration of 3 to 11 hours per day. Neurological signs, ABR, CT/MRI, EEG and the grade of the vegetative syndrome were estimated before and after SCS. In the course of SCS, 2 of the 6 patients recovered from the vegetative syndrome. Both had a localized lesion in the brain stem without a cerebral lesion on CT/MRI, with bilateral appearance of the fifth peak with prolonged latency and decreased amplitude of main peaks on ABR. The other 4 patients showed little or no improvement. They all had diffuse cerebral atrophy or low density areas on CT and almost normal ABR. One of these patients, who suffered a cerebral contusion leading to transtentorial herniation with unilateral cerebral contusion on CT and unilateral disappearance of the fifth peak on ABR, showed no recovery from the vegetative syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)