An attempt at intracytoplasmic sperm injection of frozen-thawed minke whale (Balaenoptera bonaerensis) oocytes.

Little is known about the characteristics of fertilisation events in minke whales. Cryopreserved minke whale oocytes and spermatozoa do not fertilise in a standard IVF. This study was conducted to investigate the pronucleus formation ability of cryopreserved minke whale oocytes and their subsequent development following intracytoplasmic sperm injection (ICSI). In experiment 1, frozen-thawed minke whale immature oocytes were cultured for in vitro maturation (IVM) in a maturation medium (TCM199) supplemented with either porcine follicle stimulating hormone (pFSH)/estradiol-17beta (E2) or pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG). After 120 h of IVM, oocyte survival was examined before ICSI, and showed no significant difference in morphological normality (24-36%) between the two IVM media. Two-cell embryos (two oocytes from 21 sperm-injected oocytes) were obtained when the maturation medium was supplemented with pFSH/E2 or PMSG/hCG. In experiment 2, cryopreserved maturing oocytes were investigated for the effects of repeat-culture (2 h or 24 h) on survival before ICSI. Pronuclear formation and development were examined for the effects of sperm pretreatment with dithiothreitol (DTT) and oocyte activation with ethanol at ICSI. A frequency of 49-69% of frozen-thawed maturing oocytes was used for ICSI. Although oocyte activation did not produce a significant difference in survival, pronucleus formation and embryonic development, 2- and 4-cell cleaved oocytes were observed after injection of sperm pretreated with DTT.

An experimental study of the effects of nerve root retraction on the posterior ramus.

STUDY DESIGN: The histologic and ultrastructural changes in the posterior ramus after posterior lumbar surgery were studied in rabbits. OBJECTIVE: To investigate the structural changes in the posterior ramus after posterior lumbar surgery that may cause injury to the posterior ramus after the procedure. SUMMARY OF BACKGROUND DATA: Investigators in previous studies have pointed out that low back discomfort after lumbar discectomy relates to neurogenic changes and/or myogenic changes of paravertebral muscle. However, no previous study has demonstrated the effects of excessive nerve root retraction on spinal posterior rami. METHODS: Eighteen male Japanese White rabbits were used. The posterior ramus arising from the S1 nerve root was examined after exposure of the lamina only, fenestration, or retraction of the S1 nerve root, with light microscopy and transmission electron microscopy at 2, 4, and 6 weeks after the procedure. Results were compared with a those in control specimens that did not undergo the procedure. RESULTS: In the exposed group, no distinct difference was found compared with the control specimen. In the fenestration group, especially at 6 weeks, some attenuation and splitting of myelin sheaths was observed. In the retraction group, the structural alteration was most severe. Even at 2 weeks, fragmentation of many myelin sheaths was detected. Examination of specimens by electron microscopy indicated phagocytosis of myelinated fibers at 4 and 6 weeks. CONCLUSIONS: Findings showed that posterior lumbar procedures, including retraction of paravertebral muscle, fenestration of the lamina, and retraction of the nerve root affect the posterior ramus. Excessive retraction of the nerve root has an especially disastrous effect on the posterior ramus. Such a violent maneuver within the spinal canal must be avoided.

Epstein-Barr virus infection resembling autoimmune hepatitis with lactate dehydrogenase and alkaline phosphatase anomaly.

A 73-year-old man had fever, lymphadenopathy, granulocytopenia, thrombocytopenia, ascites, pleural effusion, liver injury, and an allergic-like skin rash. Autoantibodies, such as anti-nuclear antibody, were shown, and there were lactate dehydrogenase and alkaline phosphatase anomalies and platelet-associated IgG. His liver injury resembled that in autoimmune hepatitis. He was diagnosed with Epstein-Barr virus (EBV) infection associated with autoimmunization because of his clinical course, fluctuation of anti EBV antibodies and positive EBV genome in circulating lymphocytes and serum. This case suggests a close relationship between EBV infection and autoimmunization or autoimmune-like hepatitis.

Horseshoe crab hemocyte-derived antimicrobial polypeptides, tachystatins, with sequence similarity to spider neurotoxins.

Antimicrobial peptides, named tachystatins A, B, and C, were identified from hemocytes of the horseshoe crab Tachypleus tridentatus. Tachystatins exhibited a broad spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi. Of these tachystatins, tachystatin C was most effective. Tachystatin A is homologous to tachystatin B, but tachystatin C has no significant sequence similarity to tachystatins A and B. Tachystatins A and B showed sequence similarity to omega-agatoxin-IVA of funnel web spider venom, a potent blocker of voltage-dependent calcium channels. However, they exhibited no blocking activity of the P-type calcium channel in rat Purkinje cells. Tachystatin C also showed sequence similarity to several insecticidal neurotoxins of spider venoms. Tachystatins A, B, and C bound significantly to chitin. A causal relationship was observed between chitin binding activity and antifungal activity. Tachystatins caused morphological changes against a budding yeast, and tachystatin C had a strong cell lysis activity. The septum between mother cell and bud, a chitin-rich region, was stained by fluorescence-labeled tachystatin C, suggesting that the primary recognizing substance on the cell wall is chitin. As horseshoe crab is a close relative of the spider, tachystatins and spider neurotoxins may have evolved from a common ancestral peptide, with adaptive functions.

[The effects of identity of facial expression and person on face recognition: a study using priming paradigm].

Three experiments investigated the effects of identity of facial expression and person of two successively presented faces on face recognition. In each experiment, a combination of a familiar/unfamiliar person with neutral/smile expression was presented. Four different groups of 8 or 12 undergraduates were asked to judge the facial expression or the familiarity of the second face. The results showed that reaction times in both judgments were shorter when the same person repeatedly appeared than when two different people were presented. This repetition effect was not affected by facial expression of the stimulus for expression judgments, while it depended on the expression and familiarity of the first and second faces for familiarity judgments. In facial expression judgments, reaction times to smile faces were shorter than those to the neutral faces, only when subjects were familiar with those faces. This facial expression effect appeared only when the identical familiar and smile person was repeatedly presented for familiarity judgments. These results suggested the interdependency between analysis of facial expression and that of person information in face recognition processes.

Spontaneous intramural hematoma localized in the proximal esophagus: truly "spontaneous"?

After a usual meal, a 57-year-old woman with normal hemostasis experienced a hematoemesis. She was then diagnosed endoscopically as having an intramural hematoma of the esophagus, which ranged from 18 cm to 24 cm from the incisors. The hematoma is considered to have developed not "spontaneously" but as a result of direct abrasive trauma by foodstuffs. The authors think it appropriate to use the term "spontaneous" only when the development of hematoma is unrelated to emetogenic events, impaired hemostasis, and food-induced injury.

Coexisting primary early gastric plasmacytoma and sarcoidosis with hypercalcaemia.

We report on a 61-year-old woman with coexisting early stage primary gastric plasmacytoma and sarcoidosis with hypercalcaemia. Laboratory data on admission showed hypercalcaemia, with 12.8 mg/dl, parathyroid hormone-related peptide (PTHrP) 1.2 pmol/l, C-PTHrP 69.5 pmol/l, and 1,25-dihydroxyvitamin D3 46.7 pg/ml. Neoplastic plasma cells proliferated in the propria mucosa of the stomach, showed a monoclonal immunoglobulin of cytoplasmic IgA (lambda light chain) and were positive for leucocyte common antigen and epithelial membrane antigen on paraffin section prepared from a stomach biopsy specimen. Russel bodies were present, as were crystals. Abundant sarcoid granulomas were observed in many of the regional lymph nodes around the stomach and in the dermis of a skin nodule. The patient underwent subtotal gastrectomy with administration of antimyeloma chemotherapy. We suggest that the hypercalcaemia in this patient was due to PTHrP production by neoplastic plasma cells.

Effect of interferon on GB virus C and hepatitis C virus in hepatitis patients with the co-infection.

Of 74 patients who were infected with hepatitis C virus (HCV) and received interferon, 12 (16%) were positive for RNA of GB virus C (GBV-C). RNA of GBV-C was determined in sera from the co-infected patients retrospectively, and the effect of interferon on GBV-C was compared with that on HCV in them. Titers of both GBV-C and HCV RNAs decreased during interferon in all of them. Two patients lost both GBV-C and HCV RNAs and remained clear until 6 months after treatment with interferon, while 2 lost RNA for GBV-C only and 2 for HCV RNA alone. Low pretreatment RNA titers of GBV-C and HCV correlated with the efficacy of interferon in clearing. Alanine aminotransferase returned to normal only in the patients who lost HCV RNA, regardless of the persistence or loss of GBV-C RNA. These results indicate that the response to interferon of GBV-C is comparable to but independent of that of HCV and that the persistence of GBV-C would not prevent the normalization of aminotransferases in response to interferon in patients with chronic hepatitis C.

Breast carcinoma with myeloid metaplasia--a case report.

A 49 year-old female had been suffering from primary myelofibrosis since February 1987 without receiving any treatment. In 1994, a breast mass was detected. Breast tumor biopsy revealed tubular carcinoma with intraductal components and multinucleated giant cells in the loose and myxoid stroma. The giant cells were thought to be megakaryocytes because both Factor VIII and platelet glycoprotein GP IIIa were detected in their cytoplasm. While additional mastectomy specimens and the axillary lymph nodes also revealed prominent myeloid metaplasia, there was no proliferation of the cancer cells. Granulocytic series stained for chloroacetate esterase and very few erythrocytic series were observed. This is the first case in which breast carcinoma and myeloid metaplasia coexisted in the same breast tumor.

Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity.

Small granules of horseshoe crab hemocytes contain two known major antimicrobial substances, tachyplesin and big defensin (S5), and at least five protein components (S1 to S6), with unknown functions. In the present study, we examined the biological properties and primary structure of a small granular component S2, named tachycitin. This component was purified from the acid extract of hemocyte debris by two steps of chromatography. The purified tachycitin was a single chain protein with an apparent M(r) = 8,500 on Tricine-SDS-polyacrylamide gel electrophoresis. Ultracentrifugation analysis revealed tachycitin to be present in monomer form in solution. Tachycitin inhibited the growth of both Gram-negative and -positive bacteria, and fungi, with a bacterial agglutinating property. Moreover, tachycitin and big defensin acted synergistically in antimicrobial activities. The amino acid sequence and intrachain disulfide bonds of tachycitin were determined by amino acid and sequence analyses of peptides produced by enzymatic cleavages. The mature tachycitin consisted of 73 amino acid residues containing five disulfide bonds with no N-linked sugar. A cDNA coding for tachycitin was isolated from a hemocyte cDNA library. The open reading frame coded for an NH2-terminal signal sequence followed by the mature peptide and an extension sequence of -Gly-Arg-Lys at the COOH-terminus, which is a putative amidating signal. The COOH-terminal threonine amide released after digestion of tachycitin with lysylendopeptidase was identified. The NH2-terminal 28 residues of tachycitin shows sequence homology to a part of chitin-binding regions found in antifungal chitin-binding peptides, chitin-binding lectins, and chitinases, all of which have been isolated from plants. Tachycitin showed a specific binding to chitin but did not bind with the polysaccharides cellulose, mannan, xylan, and laminarin. Tachycitin may represent a new class of chitin-binding protein family in animals.

[Interrelationship between the facial expression and familiarity: analysis using spatial filtering and inverted presentation].

We investigated interrelationship between the facial expression and familiarity using spatial filtering and inverted presentation. The stimuli were facial images (smiling and neutral faces) band-pass filtered in the spatial domain. If the filtering is carried out at low spatial frequencies, the images convey more global features than the local ones, whereas the local features are emphasized in the images filtered at high frequencies. The task of subjects was to judge the expression (Experiment 1) or familiarity (Experiment 2) of such images, which were presented in the upright or inverted orientation. The following results were obtained. 1) Spatial frequency components in the middle range (12.4 and 24.8 cycles/face-width) were important in recognizing both expression and familiarity, 2) there was close relationship between the facial expression and familiarity, and 3) smiling faces were harder to recognize when they are shown in the inverted orientation. These results suggest that there are the common and different recognition processes between facial expression and familiarity.

Complement receptor type 1 (CR1) deficiency on neutrophils in myelodysplastic syndrome.

We present a patient with myelodysplastic syndromes (MDS) whose neutrophils exhibited defective expression of complement receptor type 1 (CR1). A 73-year-old man was admitted with an evolution of MDS from RA into RAEBT according to the FAB classification of MDS. The neutrophil alkaline phosphatase (NAP) score was zero. The surface expression of membrane effector molecules on neutrophils was determined by indirect immunofluorescence using flow cytometry and monoclonal antibodies. The expression of CR1 on neutrophils as identified by staining with CD35 was defective in the patient, and the expression of other complement receptors (CR3 and CR4), Fc receptors and adhesion molecules was normal. CR1 deficiency and defective NAP score on neutrophils in the patient might account for impairment of common storage pool, presumably novel intracellular secretory vesicles.

Hepatitis C virus RNA and antibodies among blood donors in Beijing.

Blood units from voluntary as well as commercial donors in Beijing, China, were tested for hepatitis C virus RNA and antibodies, and for serological markers of hepatitis B virus infection. HCV RNA was detected less frequently in 1909 voluntary donors (5 (0.3%)), than in 1017 commercial donors (58 (5.7%)) (p < 0.001). Antibody to hepatitis C virus was detected by the second-generation enzyme immunoassay in 55 (87%) of 63 blood units with viremia. Evidence of present or past infection with hepatitis B virus was common both in voluntary (43.9%) and commercial (46.4%) donors. There were eight (13%) sera with HCV-RNA in which hepatitis C virus antibodies were not detectable by second-generation enzyme immunoassay. Of 63 HCV-RNA samples from donors, 33 (52%) were of genotype II, 18 (29%) of III and one (2%) of II + III. HCV-RNA in the remaining 11 (17%) were not classifiable into any of the genotypes I, II, III, IV and V. Genotype II was more frequent in viremic donors with elevated alanine aminotransferase levels (13/18 or 72%) than in those with normal levels (20/45 or 44%). These results indicate a low prevalence of hepatitis C virus infection in the general population in Beijing, and the limitations of identifying sera with viremia by second-generation enzyme immunoassay.

[Sequential monitoring of various HCV-related markers in chronic hepatitis C with interferon treatment].

Interferon (IFN) which is a powerful antiviral cytokine is capable to cure approximately 40% of patients with chronic hepatitis C. We should like to know in detail whether IFN is operating enough to improve chronic hepatitis C during IFN treatment. Therefore it was evaluated what kind of HCV related markers were important to monitor the clinical course. We discussed HCV-RNA (RT-PCR, DNA probe assay) and some antibodies to HCV including our new IgM antibody to a HCV core peptide, cp14 (amino acid 5-40 of the core protein). HCV-RNA assayed by RT-PCR was considered best to monitor the therapeutic effect of IFN on chronic hepatitis C. However, as it is still not in general, IgM anti-cp14 might be an alternative in monitoring the response to IFN in patients with chronic hepatitis C.

IgM antibody to a hepatitis C virus core peptide (CP14) for monitoring activity of liver disease in patients with acute or chronic hepatitis C.

Antibodies to the hepatitis C virus (HCV) core of various immunoglobulin classes were determined by enzyme immunoassays with three synthetic peptides, CP14 (amino acids 5-40 of the core protein), CP10 (5-23), and CP9 (39-74). In 135 patients with chronic type C liver disease, anti-CP14, anti-CP10, and anti-CP9 of IgG class were detected in 99%, 94%, 82%, respectively; those of IgM class in 86%, 69%, and 39%; and those of IgA class in 56%, 40%, and 4%. Thus anti-CP14 was more prevalent than anti-CP10 or anti-CP9 in every immunoglobulin class. The prevalence of IgM anti-CP14 was much higher (P < 0.001) in patients (116/135 or 86%) than in asymptomatic carriers of HCV (13/39 or 33%). In seven patients with acute hepatitis C, IgM anti-CP14 continued to decrease in two in whom hepatitis resolved, but increased in five in whom hepatitis once resolved and then exacerbated. IgM anti-CP14 was followed in 30 patients with chronic hepatitis C during 24 weeks while they received recombinant interferon alpha-2a. IgM anti-CP14 decreased remarkably within 8 weeks in all of them. Thereafter, it continued to decrease in nine patients who responded to interferon and lost HCV RNA from circulation, but started to increase in five non-responders who continued to have high titers of HCV RNA. In the remaining 16 patients in whom HCV RNA decreased once and then increased, IgM anti-CP14 continued to decrease till 20 weeks and then increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotype dependence of hepatitis C virus antibodies detectable by the first-generation enzyme-linked immunosorbent assay with C100-3 protein.

Hepatitis C virus (HCV) samples in 155 sera, from patients with chronic non-A, non-B liver disease and blood donors, were grouped into four genotypes (I, II, III, and IV) by amplification of core-gene sequences by polymerase chain reaction with type-specific primers. HCV genotypes were compared with various HCV-associated antibodies detectable by the first-generation ELISA (ELISA-1) with C100-3 protein and a second-generation immunoblot assay with four recombinant HCV proteins. Antibodies to C100-3 protein and those to its subsequence (5-1-1) were detected in 13 (93%) and 12 (86%), respectively, of 14 sera with genotype I HCV; 56 (79%) and 58 (82%) of 71 sera with genotype II; 13 (34%) and 6 (16%) of 38 sera with genotype III; and 11 (34%) and 4 (13%) of 32 sera with genotype IV. Amino acid sequences of C100-3 of genotype I HCV are conserved by approximately 90% in genotype II, but only by approximately 75% in genotypes III and IV. The sensitivity of ELISA-1, therefore, would be influenced by heterogeneity in C100-3 sequences of different genotypes.