[Talks on evaluation of students' writing (discussion)].

We talked about the evaluation of students' writing; this is thought to be difficult for teachers. We have a class "history of medicine" for freshmen in the second semester of the medical course. Students give lecture by themselves on famous historical persons in medicine such as Hippocrates, Vesalius, Pasteur and Sackett, referring to their historical significance in present medicine. As the final work of the subject they write report on "clinical medicine or medical researches 20 years in future and myself". We talked generally about educational evaluation in various aspects, concerning the methods for writing. We discussed mainly on two themes--ability to write and originality in thinking. We give a larger point to the originality, which correlates to ability to write, though we feel difficult in evaluating students' originality only by reading their reports. We also emphasize the importance of active interaction between students and teachers, and to evaluate educational activities of teachers in Japanese universities.

[An autopsy case of an infant born with a caul who died due to asphyxia after live-birth].

We reported an autopsy case of a child born with a caul. The infant was a female and seemed to be full-term. No remarkable pathological findings for disease or anomalies were observed. The floating test of lungs was partially positive. And on the gastro-intestinal floating test, only stomach floated. In histological examination, the alveoli in the lungs were partially slightly open. We thought that those were due to the cardio-pulmonary resuscitation. Pulmonary surfactant was positive immunohistochemically. And the stable microbubble test on the liquid in her stomach was very strong. Even if a child is born alive, he or she will soon die due to asphyxia but for the production of surfactant in the lungs. We can evaluate more precisely the potential respiratory ability of a neonate died due to asphyxia by means of stable microbubble test on newborn gastric contents and immunohistochemical staining of surfactant in lungs.

[Bruise-like discolorations can appear after death].

It is not sufficiently emphasized in our country that bruising can also appear post-mortem. We report two cases in which we observed discolorations which looked like ante-mortem bruising. Case 1: A 37-year-old man was found prone on a river shore and taken out of the water by a rescuer by grasping at the right upper arm approximately one hour and 30 minutes after death. At inspection, two thumb-sized discolorations resembling ante-mortem bruising were observed on the lateral and frontal surfaces of the right upper arm. Case 2: A 40-year-old woman was found prone immersed in a moat and taken out of the water in the above-mentioned manner approximately one hour and 45 minutes after death. At inspection, two thumb-sized discolorations appearing to be ante-mortem bruising were observed on the inner surface of the right upper arm. The cause of death in both instances was drowning. Bibliographic investigation revealed that bruising of significant size can appear after death. We speculate generally on conditions for generation of post-mortem bruising. Additionally, we believe that post-mortem bruising should be sufficiently considered, because it can be important whether a person was grasped when he or she was alive or dead.

Non-proteolytic activation of human prorenin by anti-prorenin prosegment (pf#1: 1P-15P) antiserum.

Recombinant human prorenin was activated by incubation with anti-prorenin prosegment (L1PPTDTTTFKRIFLKR15P) antiserum at 4 degrees C. This activation was dependent on the concentration of the antiserum and incubation time. After the activation no molecular weight alteration of prorenin was observed by immunoblotting analysis. A peptide of L1PPTDTTTF8P as well as L1PPTDTTTFKRIFLKR15P potently interfered with the activation. Most of the activated prorenin bound to Protein A Sepharose CL 4B. The Km and Vmax values of the activated prorenin were 0.2 microM and 23.7 micrograms Ang I/ml/h, respectively, which were similar in level to those of mature renin obtained by trypsinization.

[Investigating methods for age estimation (1). Height of the apex of the medullary cavity of the humerus].

The relationship between age and the height of the proximal apex of the medullary cavity of humerus was investigated on 13 male bodies (29-62 years) and 12 female bodies (22-80 years) and a provisional criterion for age estimation was obtained. When the apex is (1) at a height of the surgical neck, the estimated age is under 50 years in both sexes, (2) at the epiphyseal line, over 50 years in males, and from 50 to 75 years in females. In a few cases, however, the apex reaches the line even in the very young age group (20-30 years). (3) When a cavity or rough structure is present above the line in addition to phase (2), the estimated age is over 75 years in females, but unknown in males. We also discuss the validity of Koshinaga's criterion (1951).

Estimation of stature from somatometry of skull.

In order to investigate the possibility of estimating stature from somatometry of the skull, we carried out a study on 124 Japanese cadavers (comprising 77 males and 47 females) that had been autopsied at our laboratory between July 1986 and June 1991. Somatometry of the skull was performed on diameter (distance between glabella and external protuberance) and circumference (length around the skull through two points: the glabella and the external protuberance). The regression equations calculated were as follows: stature in males = (diameter + circumference) x 1.35 + 70.6 (standard error of estimate (S.E.) = 6.96 cm); stature in females = circumference x 1.28 + 87.8 (S.E. = 6.59 cm); stature in both sexes = (diameter + circumference) x 1.95 + 25.2 (S.E. = 7.95 cm). These S.E.s appear to be larger than those obtained for other parts of the body. However, in cases where identification is required by means of only the skull, this method could prove useful.

Formation of the chordamesoderm in the amphioxus embryo: Analysis with Brachyury and fork head/HNF-3 genes.

The embryonic development of amphioxus (cephalochordates) has much in common with that of vertebrates, suggesting a close phylogenetic relationship between the two chordate groups. To gain insight into alterations in the genetic cascade that accompanied the evolution of vertebrate embryogenesis, we investigated the formation of the chordamesoderm in amphioxus embryos using the genes Brachyury and fork head/HNF-3 as probes. Am(Bb)Bra1 and Am(Bb)Bra2 are homologues of the mouse Brachyury gene isolated from Branchiostoma belcheri. Molecular phylogenetic analysis suggests that the genes are independently duplicated in the amphioxus lineage. Both genes are initially expressed in the involuting mesoderm of the gastrula, then in the differentiating somites of neurulae, followed by the differentiating notochord and finally in the tail bud of ten-somite stage embryos. On the other hand, Am(Bb)fkh/HNF3-1, an amphioxus (B. belcheri) homologue of the fork head/HNF-3 gene, is initially expressed in the invaginating endoderm and mesoderm, then later in the differentiating notochord and in the tail bud. With respect to these two types of genes, the formation of the notochord and tail bud in amphioxus embryos shows similarity and dissimilarity with that of the notochord and tail bud in vertebrate embryos.

Duplication of an amphioxus myogenic bHLH gene is independent of vertebrate myogenic bHLH gene duplication.

Gene duplication is thought to be a major genetic change that may have permitted the evolution of vertebrates from invertebrates. The myogenic genes encode basic helix-loop-helix (bHLH) transcriptional factors essential for the formation of skeletal muscle. The invertebrate genome contains only a single myogenic bHLH gene, whereas the vertebrate genome contains four (MyoD, Myf-5, myogenin and MRF4). Since the tunicate genome contains a single myogenic bHLH gene, its duplication might have occurred some time during chordate evolution. To determine whether the duplication of the myogenic bHLH gene occurred prior to, or after the divergence of vertebrates from the cephalochordate lineage, we amplified target fragments from the amphioxus, Branchiostoma floridae, by means of PCR. Sequence analysis and genomic Southern analysis revealed that the amphioxus genome contains two myogenic bHLH genes (BMD1 and BMD2). A comparison of the amino acid sequences in the bHLH domain between BMD1, BMD2 and four vertebrate myogenic bHLH gene products, however, showed that neither BMD1 nor BMD2 resembled any of the four genes. These results suggested that the duplication of amphioxus myogenic bHLH gene occurred independently of that leading to the four myogenic bHLH genes in vertebrates.

[Relationship among "kenshi" (inquest), "kenshi" (postmortem examination) and "ken-an" (postmortem inspection) in Japan].

Inquest ([symbol: see text]) and postmortem examination ([symbol: see text]) have the same pronunciation of "kenshi" in Japan. So we are inclined to use these terms with confusion or substitution. We discussed the differences among three "kenshis" adding to postmortem inspection ([symbol: see text]; "ken-an") with investigating references. Many forensic scientists described that inquests were done by public prosecutors and inspections by medical doctors. But there were various interpretations of postmortem examination, e.g. "postmortem examination is done by public prosecutors or police", "it is the same as inspection by medical doctor", "it is one of the aids of inquest", "it is also including forensic autopsy". Although being no differences between the two kenshis written by two kinds of different Chinese characters ([symbol: see text]). This is caused an only problem in writing Japanese languages, it happens especially when we have to use Chinese characters for everyday use. In order to define this postmortem examination in Japan, we paid attention to corpse procedures. We made a figure to include each interpretation and to understand that postmortem examination had several meanings from a narrow sense to a wide sense. We suggested that the most confident definition was of the widest sense which meant the inspection and autopsy by doctors and the inspection by public prosecutors or police.

Physiological, morphological and anatomical responses of Fraxinus mandshurica seedlings to flooding.

Two-year-old Fraxinus mandshurica Rupr. var. japonica Maxim. seedlings were flooded to 8 cm above soil level for 70 days. The flooding treatment altered the growth, morphology, stem anatomy and ethylene production of the seedlings. Although flooding did not affect height growth, it stimulated diameter growth of the submerged stems by increasing both the number and size of wood fibers produced; however, the thickness of the cell walls of the wood fibers was reduced by flooding. In response to the flooding treatment, the seedlings formed abundant hyperhydric tissues, originating from the vicinity of lenticels on the surface of the flooded stems, and adventitious roots, which grew through the hyperhydric tissues. Aerenchyma tissues were observed in the bark of the adventitious roots. The flooding treatment did not affect dry weight increment of leaves and stems, but it reduced the total dry weight increment of the root system even though it promoted adventitious root formation. Flooding also enhanced ethylene production in the submerged portions of stems. The potential roles of flood-induced ethylene in cambial growth and adventitious root formation in flooded plants are discussed.

[An autopsy case of a pacemaker implanted patient].

A pacemaker implanted patient (female, 78 years old) was found dead in her bed and judicially autopsied. Before postmortem inspection the pacemaker was removed by a doctor. We report the problems in such as the doctor's treatment, additionally accommodation of medical examiners and police, and determination of a cause of death in autopsy.

[Age estimation by appearance of gray hair in pubic hair].

The pubic hair of Japanese males aged 12-90 years old (n = 123) and females 14-85 ys. (n = 80) autopsied during 1984-1993 was investigated to determine its appearance age and possible appearance age of gray hair. As the appearance degree of gray hair in pubic hair 5 stages were applied to samples to establish a criterion for age estimation. Stage 0; no gray hair, 1; 1-3 gray hairs, 2; a few-10, 3; 10 gray hairs-2/3 of the entire pubic hair, 4; more than 3/4. The appearance age of gray hair with appearance rate 100% (75%) was more than 65 (55) ys. in both sex. The possible appearance age was more than 30 ys. in males and 36 ys. in females, practically younger than "45 years old" which had been described in texts. The age range with a hit rate 100% is at stage 0; less than 62 ys. in males, less than 59 ys. in females, at stage 1; 30-64 ys. in males, 36-60 ys. in females, at stage 2; 43-72 ys., 39-85 ys. and at stage 3; 49-77 ys,--.

[Cultural background of the Japanese bioethics].

We attended the 11th Liaison Society of Ethics Committees in Medical Schools in Japan. Three symposiums were held under the themes of quality of life (QOL), stopping medical cure and the Japanese bioethics. Symposists were medical practitioners, teaching staffs in universities and a person of religion. In the first symposium, the definition of QOL, the usage of the term and the method of its evaluation were discussed. In the second symposium, an internist and a neonatologist reported several cases and stated problems and countermeasures in terminal care in cases that they could not maintain QOL. A person of religion made his opinion on the problems. In the final symposium were stated Japanese bioethics from the aspects of ethics and cultural anthropology. They emphasized differences in bioethical view between the Japanese and the Europeans and Americans, and a need to reform medical education in Japan. It is difficult to define QOL and to care patients at terminal stage, because present-day persons have various senses of value. Especially, Japanese have taken Western culture into our traditional social structures with its original style. Therefore, we have dual culture, as recognized in communication. Although it is very important to communicate sufficiently between patients and doctors, we consider that the dual communication has interrupted their mutual understandings. Incidentally, Western medicine had originally dual structure of art and technology. But we have taken only the technological aspect. That is probably the reason why human relations have been getting worse. It would be necessary for us to attend to these two dual structures in order to solve bioethical problems in Japan.

[A case of examination of skeletal remains--how many bodies did they come from?].

We examined skeletal remains, with the main intention of estimating how many bodies they had come from. The samples were a skull with defect of its base, an upper jaw, two lower jaws (No. 1, No. 2) and two skeletal bodies (No. 1: with no skull, No. 2: consisting mainly of the lower extremities). In examining the mutual relationship among them, we utilized adjustability between the skull and the lower jaws at the temporomandibular joint, and between the lower jaws and the upper jaw in the biting manner of their teeth. We concluded that the most probable combination was of two bodies, i.e. [skull + lower jaw No. 1 + skeletal body No. 1] and [upper jaw + lower jaw No. 2 + skeletal body No. 2]. Beside the above presentation, we did comment on several problems in personal identification and estimation of postmortem interval.

Effects of flooding on leaf dynamics and other seedling responses in flood-tolerant Alnus japonica and flood-intolerant Betula platyphylla var. japonica.

Two-year-old seedlings of Alnus japonica Steud. and Betula platyphylla var. japonica Hara were flooded from mid-June to early November, to study the effects of flooding on seedling survival and growth, morphological changes in stems and roots, leaf emergence, leaf fall, and leaf longevity. In A. japonica, growth was not affected by flooding, except for a slight decrease in height growth, but some morphological changes of stems and roots were observed, i.e., stem base hypertrophy, hypertrophied lenticels, formation of adventitious roots and development of new roots. In B. platyphylla var. japonica, growth was severely reduced by flooding and all seedlings died by the 20th week of flooding, without showing any adaptive morphological changes in stems or roots. Flooding induced rapid depression of leaf emergence, promoted leaf abscission, and reduced leaf longevity in B. platyphylla var. japonica. In contrast, in A. japonica, basal leaf senescence was delayed in flooded seedlings, thereby extending leaf longevity compared with unflooded seedlings.

Estimation of time of death by quantification of melatonin in corpses.

A method for the estimation of time of death (TOD), was evaluated by measuring the melatonin (MT) content of pineal bodies (PBs), sera and urine samples from 85 cadavers. A total of 44 cadavers were investigated in Sapporo (geographical coordinates N 43 degrees 4', E 141 degrees 21') and 41 in Tokyo (N 35 degrees 39', E 139 degrees 44'). MT contents were measured by radioimmunoassay (RIA) in 75 PBs, 27 sera and 14 urine samples. Exponential differences of pineal MT content were recognized between peaks in nighttime and nadirs in daytime, ranging from 0.099 to 63.2 ng/PB. Circadian rhythms were also observed for the concentrations of MT in serum (11-205 pg/ml), and in urine (7.5-137.5 pg/ml). Consequently, criteria for the TOD estimation are proposed as follows. 1) Pineal MT contents--(1) 0-0.2 ng/PB: TOD 1100-1700 hours, (2) 0.2-0.3 ng/PB: TOD 0700-2000 hours, (3) 0.3-1 ng/PB: inconclusive, (4) 1-4 ng/PB: TOD 1600-1000 hours, (5) 4-8 ng/PB: TOD 2000-0800 hours, (6) over 8 ng/PB: TOD 2000-0500 hours, 2) Serum MT concentration--(1) 0-100 pg/ml: inconclusive, (2) over 100 pg/ml: TOD 2200-0100 hours, and 3) Urinary MT concentration--(1) 0-35 pg/ml: inconclusive, (2) over 35 pg/ml: TOD 1800-0600 hours. The range of the estimation can be limited by a combination of these 3 criteria. The present method can be combined with other methods for estimating the TOD to decrease the range.

[Estimation of age from epiphyseal union degrees of the sternal end of the clavicle].

The sternal ends of right clavicles of Japanese aged 13-31 years (n = 54) autopsied during 1982-1992 were observed for determining degree of epiphyseal union to establish a criterion for age estimation. As the degree of union 5 stages (McKern and Stewart, 1957) were applied to samples: Stage 0; without any union between the trunk and the epiphyseal cap, stage 1; between stage 0 and stage 2, stage 2; half united, stage 3; between stage 2 and stage 4, and stage 4; completely united. As results, in females union appears to proceed faster than in males. There were cases in which union reached stage 4 in younger ages. As a criterion for age estimation it is proposed at present that in both sex, when union is in stage 0, age is certainly estimated to be less than 22 years old and in stage 4, more than 19 years old. Comparing the present data with the investigation about American males by McKern and Stewart (1957), in Japanese males union proceeds more fast than them. The authors realized that the correct criteria should be used in age estimation on a sample belonging to the same race and to the times. The clavicles in skeletal remains tend to be preserved outdoors and are suitable for estimating age from adolescence to about 30 years old. Therefore the present criterion would be very useful in districts such as Hokkaido where skeletal remains are commonly found.

Production of monoclonal antibodies against diquat and its enzyme-linked immunosorbent assay.

Monoclonal antibodies(MoAbs) very specific to diquat (DQ) were produced. An immunogen was synthesized by binding DQ to bovine serum albumin via a diazo-coupled derivative. BALB/c mice were immunized i.p. monthly with 0.25mg of the immunogen for five months. Their spleen cells were fused with P3U1 myeloma cells and hybridoma clones secreting MoAbs were obtained. Two MoAbs were selected and subtyped to be IgM and IgG3. The MoAbs recognized DQ but did not bind to paraquat and other analogues at all. A datum obtained from a clinical sample demonstrates that an enzyme-linked immunosorbent assay system using one of the MoAbs is useful in the practice of toxicological analysis.