The causes of perinatal mortality in Bulawayo, Zimbabwe. The causes of all perinatal deaths at Mpilo Maternity Hospital were investigated over a 12-month period, during which there were a total of 466 stillbirths and 379 neonatal deaths, with a perinatal mortality rate of 36.0/1000 births in Bulawayo, Zimbabwe. The causes of death were in order of importance; congenital syphilis (20.5 pc), birth asphyxia (18.8 pc), unexplained stillbirths (11.8 pc), hyaline membrane disease (11.5 pc) neonatal septicaemia (10.8 pc), congenital malformations (7.7 pc), pregnancy induced hypertension (5.4 pc), placental abruption (4.9 pc), congenital infection (2.2 pc) and other causes (6.4 pc). Eleven pc of mothers booking in antenatal clinics had positive syphilis serology. Most were successfully treated. But over 400 mothers with early syphilis escaped treatment usually because they booked late or failed to book at all at antenatal clinics (74 pc) and occasionally because they had false negative results or were infected after early booking (27 pc). They delivered 101 stillbirths, most of whom died prematurely before labour and often had abdominal distension. There were 72 neonatal deaths, most of whom were preterm babies with respiratory distress and often hepatosplenomegaly. One half of the deaths from asphyxia were caused by prolonged obstructed labour and one quarter by prolapsed cord, stuck head in breech delivery and retained second twin. The incidence of both early and late onset neonatal septicaemia was very high with Group B Streptococci, Kliebsiella and Staphylococcus aureus the predominant pathogens. Improved antenatal, intrapartum and neonatal care could substantially reduce the perinatal mortality rate by preventing congenital syphilis and birth asphyxia and by treating hyaline membrane disease and neonatal septicaemia.


The immunoglobulin levels in infants attending a routine health clinic at Mbare Polyclinic. This study establishes the reference ranges for immunoglobulins in health infants attending a City Health Polyclinic. There were sex differences in the immunoglobulin level (p < 0.05). A wide range of immunoglobulins concentrations were apparent. This was consistent with other reports. In addition, an age dependency in the immunoglobulin levels was also observed. This study is constituted in the understanding of immunoglobulin pathology.


The molecular epidemiology of rotavirus infection in Ga-Rankuwa, southern Africa. Rotaviruses were detected in 32.8 pc (96/292) of stool specimens collected from infants and young children with gastroenteritis attending the rehydration unit at Ga-Rankuwa Hospital between January and December 1989. A seasonal distribution was observed with an increase in numbers shedding the virus during the colder months of the year. Based on variations in the migration pattern of the RNA genome segments when passed through polyacrylamide gels, seven different RNA electrophoretypes were identified; 82 pc of the patients had virus with long patterns and 17 pc with short patterns. A mixed infection was observed in one case with both a long and a short virus. A single dominant long electrophoretype persisted throughout the 12 month study period, whereas the other minor types co-circulated at varying intervals.


Antimicrobial activity of essential oil from Hoslundia opposita. The essential oil from the leaves of Hoslundia opposite was extracted by hydrodistillation. GC/MS analysis of the volatile oil showed that it contains largely the sesquiterpenes and sesquiterpene alcohols. The essential oil was tested against eight different bacterial species and one fungal species. The antibacterial and antifungal properties of the essential oil from Hoslundia opposite were determined by using seeded agar plates with wells into which was placed the oil, and flasks of yeast extract and sucrose broth for mycelial growth of Aspergillus niger. After incubation for 24 hours, the diameter of the inhibition zone was measured for the antibacterial tests and after seven days, the dry weight of the mycelia was measured and a percentage of inhibition calculated using controls where no samples were added. The results obtained showed that the essential oil from this plant has significant activity against Aspergillus niger, Acinetobacter calcoacetica, Brochothrix thermosphacta and Flavobacterium suaveolens. These were most affected by the volatile oil from Hoslundia opposita.


Development of Health Systems Research and National HSR networking in Zimbabwe. Health Systems Research (HSR) aims at improving the quality of health care delivery through health managers with a management tool for decision making. Since 1988, over 100 health cadres from provincial and district health teams, central hospitals and municipalities have received training in HSR methodology. This training has resulted in the implementation of 12 research projects with the results being used to solve immediate problems identified. The development of HSR in Zimbabwe has focused on training priority setting for essential health research and the development of infrastructure and local resource groups to sustain the initiative. The HSR unit in conjunction with the Medical Research Council of Zimbabwe (MRCZ) have developed a computerised databank for keeping track of all health and medical research conducted in the country. The databank is a mechanism for co-ordinating and promoting research through minimising duplication of research effort in the dissemination and utilisation of research findings. Prospects for HSR in Zimbabwe are good; further development requires adequate funding, networking of all research groups and a national commitment to build a critical mass of trained health research manpower at all levels.


Asymptomatic significant bacteriuria among pregnant and non-pregnant women in Sagam, Nigeria. In a study carried out among 510 pregnant and 304 non-pregnant women at Ogun State University Teaching Hospital, Sagamu, 122 (23.9 pc) of the pregnant women and 37 (2.2 pc) of the non-pregnant women had significant bacteriuria. The rate amongst the pregnant women was significantly higher than in non-pregnant women (P < 0.001). There was a correlation between pyuria and significant bacteriuria in 45 (8.8 pc) of the pregnant women and in 11 (3.6 pc) of the non-pregnant women. Significant bacteriuria was closely associated with nulliparae. Most of the pregnant women with significant bacteriuria belonged to the low socio-economic group. Unlike in previous studies in this country, the most prevalent organism in this study was the Staphylococcus aureus.


New trends in chloroquine efficacy in the treatment of malaria: significance of low (scanty) parasitaemia in an endemic area, with emerging chloroquine-resistant P. falciparum. In a continuous malaria therapy surveillance, using in vivo (WHO) seven-day-test, extended to 14 days follow up, we evaluated the significance of low (scanty) parasitaemia, in an area with chloroquine resistance P. falciparum (CRPF), where self-medication is widely practised. We found that 30.9 pc of the patients screened had Plasmodium species, and 71.4 pc of these had low parasite counts of less than 500 parasites/mm3, whole blood. Eight pc of these were febrile and 41.7 pc of the parasite strains were not susceptible to chloroquine. Parasite strains from four of the patients were also resistant to other antimalarials. These patients gave psychosomatic symptoms, and were seen by a psychiatrist. We conclude that 41 pc of the patients with low parasite counts consist of patients with CRPF and/or multiple-drug resistant P. falciparum in this area. These do not only cause chronic anaemia, but also may be responsible for moderate psychosomatic symptoms in all ages.


Tonometer-induced corneal injury: a report of two cases. To avoid cross-infection, tonometers are sterilised before use on patients. When not carefully handled, the sterilising solutions could cause ocular injury. Two cases of corneal damage caused by alcohol used in sterilising the Schiotz tonometer are reported. Suggestions are made on how to avoid such iatrogenic injuries.


Congenital cystic dilation of the intrahepatic bile ducts (Caroli's disease): report of a case and review of the literature. A case of congenital cystic dilation of the intrahepatic ducts (Caroli's disease) or type V congenital intrahepatic duct cysts, which was followed for eight years after successful surgical extirpation of the cyst is reported. The presentation and management of this relatively rare entity is discussed and there is a review of the literature.


Characterization of smooth muscle caldesmon as a microtubule-associated protein. We have previously shown that nonmuscle caldesmon copurified with brain microtubules binds to microtubules in vitro [Ishikawa et al.: FEBS Lett. 299:54-56, 1992]. To explore the role of caldesmon in the functions of microtubules, further characterization was performed using smooth muscle caldesmon, whose molecular structure and function have been best-characterized in all caldesmon species. Smooth muscle caldesmon bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with the binding constant of 1.1 x 10(6) M-1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. Partial digestion of the caldesmon with alpha-chymotrypsin revealed that the binding site of the caldesmon for microtubules lay in the 34-kDa C-terminal domain. When the caldesmon was in the dimeric form in the absence of a reducing agent, the caldesmon cross-linked microtubules to form bundles. Further, the caldesmon potentiated the polymerization of tubulin, and inhibited the in vitro movement of microtubules on dynein. These results suggest that caldesmon may be involved in the regulation by Ca2+ of the functions of microtubules.


Thrombin, epidermal growth factor, and phorbol myristate acetate stimulate tubulin polymerization in quiescent cells: a potential link to mitogenesis. Previous studies suggest that alterations in the microtubule (MT)-tubulin equilibrium during G0/G1 affect mitogenesis. To determine the effect of growth factors on the MT-tubulin equilibrium, we developed a radioactive monoclonal antibody binding assay (Ball et al.: J. Cell. Biol. 103:1033-1041, 1986). With this assay, 3H-Ab 1-1.1 binding to cytoskeletons in confluent populations of cultured cells is proportional to the number of tubulin subunits polymerized into MTs. We now show that purified alpha-thrombin increases 3H-Ab 1-1.1 binding to cytoskeletons of serum-arrested mouse embryo (ME) fibroblasts from 1.5- to 3-fold. This stimulation is dose-dependent and correlates with concentrations of thrombin required for initiation of DNA synthesis. Other mitogenic factors, epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA), also stimulate MT polymerization. Addition of colchicine (0.3 microM) eight hours after growth factor addition, blocks stimulation of 3H-thymidine incorporation by thrombin, EGF, or PMA, suggesting that tubulin polymerization or subsequent events triggered by MT polymerization are required for cells to enter a proliferative cycle. Consistent with models for autoregulation of tubulin synthesis, thrombin, EGF, and PMA all increase tubulin synthesis 9 to 15 hr after growth factor addition, raising the possibility that the decrease in free tubulin and subsequent stimulation of tubulin synthesis is linked to progression of cells into a proliferative cycle. Colchicine addition to these cells also stimulates DNA synthesis, but colchicine-stimulated cells enter S phase 6 to 8 hr later than those stimulated by growth factors. This delayed stimulation may be related to the time required for degradation of tubulin-colchicine complexes below a critical level. These data suggest that regulation of cell proliferation may be linked to increased MT polymerization and the resulting decrease in free tubulin pools.


Epidemiology of low back pain.

Epidemiology of low back pain. At present, although there have been many epidemiological studies of risk factors for low back pain, there are few risk factors established in prospective studies; and our understanding of them remains relatively crude. Individuals in jobs requiring manual materials handling, particularly repeated heavy lifting and lifting while twisting, are at increased risk of back pain leading to work absence. In addition, exposure to whole-body vibration and job requirements for static postures are associated with back pain. Individual trunk strength has not been consistently demonstrated as associated with back pain; although there is some suggestion that when work requirements for heavy lifting exceed individual capacities, back pain is more likely to occur. The pattern of peak age at onset in the 20's is consistent with back pain development early in working life. Among other individual characteristics, only cigarette smoking has consistently been associated with back pain; and the biological mechanism for this finding is not understood. Evidence with respect to spinal flexibility, aerobic capacity, educational attainment and other variables is suggestive but not consistent. There is some evidence that the individual's relation to work, expressed as job satisfaction or supervisor rating, is also related to work absence due to back pain. While it is possible to describe, however crudely, the characteristics placing people at risk for back pain leading to work absence and/or medical attention, the problem of predicting chronicity and thus identifying patients for more intensive clinical intervention remains unresolved. At this time, only age of the patient and certain clinical features of the back pain such as the presence of sciatic symptoms, duration of the current episode, and history of prior episodes are consistently demonstrated predictors. In chronic patients, there is suggestive evidence that spinal flexibility, trunk strength, and certain psychological characteristics such as coping skills, fear and avoidance of pain or movement, job satisfaction, attribution of fault and hysterical or hypochondriacal features are associated with treatment failure. In addition, there is suggestive evidence that the availability of alternative work placement may allow for earlier return to work than otherwise. While the availability of disability compensation in excess of usual wages may serve as a disincentive to return to work. The latter-cited remain to be verified, while findings in chronic patients remain to be tested in acute. Further, the role of physical demands of work in duration of back pain episodes has not been well studied.


Back pain in children and teenagers.

Back pain in children and teenagers. Controversial opinions have been published concerning the frequency of LBP among children and adolescents. Studies from orthopaedics or neurosurgical departments have reported low figures for prevalence of specific LBP due to serious disorders. Field surveys, on the contrary, have shown that cumulative life prevalence of non-specific LBP in children and teenagers can be comparable to the prevalence data for adult populations. Some specific diagnoses are more common or characteristic of children complaining of LBP. Age, gender, sports activities and family history of LBP have been found to be significantly associated with an increased prevalence in non-specific LBP among children. Low back pain among children and teenagers is common and should be recognized. This chapter provides guidelines for a clinical approach and differential diagnoses. Most back pain in these age groups is benign and should be treated as such.


Psychosocial issues in the prevention of chronic low back pain--a literature review. This chapter has reviewed research on psychological and social factors associated with the onset and progression of low back pain. From this review it can be concluded that psychosocial traits appear to be important contributors to the course of pain and disability though methodologically well-designed longitudinal studies are rare. For this reason it is difficult to assess the relative importance of, for example, psychological distress compared with work stress. Furthermore, the mechanisms by which specific variables effect back pain remain unknown. The answer, no doubt, lies in longitudinal studies which employ multicausal models. It has been noted the psychosocial treatments which have proven effective for chronic pain populations are rarely assessed with acute pain patients. Some problems are the inaccessibility of acute back pain sufferers to psychologists, the difficulty of isolating the effect of one component of a multidisciplinary programme and the lack of uniform practice of psychosocial techniques. None the less, programmes which include psychosocial interventions appear to have superior results to those which do not. Since these techniques are often simple and inexpensive to include they should be incorporated into all treatment programmes where the potential for chronic pain syndrome exists. Gaps and flaws in current research methodologies have been identified and suggestions for future investigations have been proposed. In addition we have attempted to provide some practical guidelines for health care professionals to help them identify salient psychosocial issues which may effect the course of their patient's treatment. Recommendations for assessment and referral are also provided.


Back schools in prevention of chronicity. This chapter has reviewed the role of back school and educational programmes for the common and non-specific acute and subacute low back pain patient. The following seems to come out of this review. Education is an important part of patient care. However, several questions arise about the content of the education, the selection of patients, the patient compliance to instruction given, how the information is retained, and which outcome measures should be used. It is also important to realize that the back school is a modality or a tool that may be used as an adjunct, but as a sole treatment it seems to have less impact than in combination with other structured or goal-oriented programmes. When a back school is instituted in a hospital or in industry, it requires administrative and budgetary support and a multidisciplinary staff to successfully carry out the programme. The information given must be adapted to the needs of the participants and all members of the team must give the same information to the patient. A poorly structured back school where patients are dumped because the physician or other health care provider has nothing else to offer is a poor solution for the patient, a poor solution for the health care provider, and can only increase the patient's discomfort and health care costs.


Prevention of low back pain: basic ergonomics in the workplace and the clinic. Redesigning the job is a strategy for preventing low back injuries at work or for accommodating injured employees who return to work. An evaluation of the physical job demands is necessary in either strategy. Several job demands are associated with low back pain and injury--heavy physical work, static or postural effort, dynamic work-load and exposure to wholebody vibration. Traditional work measurement studies emphasize a rigorous task analysis. By adding biomechanical, physiological and psychophysical measurements, a comprehensive evaluation is possible. There is no standard scheme for a workplace evaluation. The method depends on the end use of the analysis. Job evaluation for workplace design requires an emphasis on equipment and work conditions; evaluation for placement of injured employees should emphasize the operational demands of the tasks. Few studies considered the multifactorial aetiology of low back pain. Most studies that measured the magnitude of biomechanical, physiological and psychophysical stresses attempted to define peak work-loads. The attempt to evaluate the effects of subacute cumulative traumas is only in the beginning. Most ergonomic intervention programmes modify the loads, the design of objects handled, lifting techniques, workplace layout and task design. The effectiveness of these interventions in controlling medical costs or morbidity has not been clearly demonstrated. Consequently, occupational risk factors may be more important for evaluating disability. Job familiarity is the key to effective medical management. Ergonomic analysis procedures may be useful within rehabilitation settings that also provide placement services. The reason is that they facilitate communication between all elements involved in the rehabilitation process. Proper communication procedures are also crucial in implementing ergonomic interventions in the workplace. A health care provider should be part of a task force that oversees these interventions. Future effort should be directed to finding a method that health care practitioners could be competent to carry out effectively in a clinical setting. Expert systems offer promising results in disseminating ergonomic knowledge in primary and secondary health care facilities.


[Nuclear size and character of the nucleolar organizer in benign and malignant follicular tumors of the thyroid gland]. Histologic sections of 11 benign and 11 malignant follicular tumours of the thyroid were compared from the point of view of subsequent practising cytologic diagnosis. Following features were analysed in 100 randomly chosen thyreocytic nuclei: maximal nuclear diameter, nucleolar organizer rates (AgNOR) inside and outside the nucleoli, total AgNOR according to a modified Crocker s et al. method, total nucleolar rate and maximal nucleolar diameter, proportion of maximal nucleolar diameter and maximal nuclear diameter, AgNOR in vicinal nontumorous thyreocytes. All of them except total AgNOR did not show any significant differences according to modified t-test. But the increase of total AgNOR in follicular carcinomas lost its significance in Duncan's test of variance analysis because of a high maximal nucleolar diameter regarding big figures in 4 carcinomas (2 of them oxyphilic celled). In most tumours AgNOR inside nucleoli could not be reliably distinguished and quantified even after shortening of silver impregnation. Mean maximal nuclear diameter bigger than 7 nm, indicating malignant follicular lesion according to literature, could not be verified in 8 tumours- it was smaller in 5 carcinomas and bigger in 3 benign tumours. Proportion of maximal nucleolar diameter and maximal nuclear diameter was stable in benign and malignant tumours as well as the AgNOR in peritumorous thyreocytes. All the analysed nuclear features are lacking in sufficient prediction value for distinction of benign and malignant follicular tumours of the thyroid.


[Preparation of bone marrow obtained by trephination biopsy]. Easy paraffine technique in processing of bone marrow samples is convenient for structural evaluation and for immunohistology as well. A short fixation in 10% formalin will do, especially when combined with decalcination in Chelaton III. Preparation of sections, staining and immunohistology are analogous to other tissues. Nevertheless, it would be better try to dilute antibodies higher than is usually recommended even if a longer incubation were needed. Intensity of staining can be increased by performing lead sulphocyanate treatment in a microwave oven.


[Evaluation of bone marrow samples obtained by trephination biopsy]. A well performed and processed trephine renders a perfect idea about tissue and cellular structure of bone marrow. Gentle taking and adequate processing of a sample are a prerequisite for successful biopsy. The most convenient and often sufficient is the paraffine technique with an entry evaluation of the first section and setling up the group diagnosis. Another time, it stimulates further choice of immunohistology and electron microscopy. There are presented detailed evaluation schemes for marrow suppression, hyperplasias, myelofibroses and tumours. They were checked in a group of 400 cases.


[Fibromatoses and related disorders in childhood]. A retrospective study of fibromatoses and related diseases was performed on a series of 34 children. Aggressive forms of fibromatoses similar to those in adults as well as typical forms of childhood fibromatoses and fibrous proliferations, such as sternocleidomastoid tumor, infantile myofibromatosis, digital fibromatosis and fibrous hamartoma were observed. Immunohistochemistry revealed muscle specific actin in eleven out of 13 cases, including hyaline cytoplasmic inclusions in digital fibromatosis. In two patients with infantile myofibromatosis a coexpression of actin and desmin was found. One of two cases of infantile type of aggressive fibromatosis was weakly actin positive whereas the other was negative. This result suggests poorly differentiated character of cells in infantile fibromatosis. Clinicopathologic correlation showed that extraabdominal fibromatoses had a strong propensity for local recurrence. Multiple lesions affecting different muscle groups were diagnosed in two boys. Abdominal fibromatosis affected two girls and two boys, in contrast to adult forms which occur exclusively in women.


[Secondary adenocarcinoma of the ovary and problems in diagnosis]. In a group of 10 secondary ovarian adenocarcinomas primary tumours were verified by biopsy in colorectal region (8 cases), once in gall bladder and once in appendix. The age of patients varied between 36 up to 60 years. The lesion was unilateral in 8 cases, bilateral in two. Six colorectal adenocarcinoma secondaries reminded a bit of endometroid carcinoma. Two colorectal and a gall bladder carcinoma secondaries resembled ovarian mucinous cystadenocarcinoma. Signet ring cells were not present. Appendical mucinous cystadenocarcinoma produced a metastasis that was alike borderline ovarian mucinous cystadenoma. Intracellular mucin reaction was negative or focally positive in former 6 cases, a diffuse positivity was found in 4 cases.


[Undetected psychiatric disorders in primary health care. A need for psychotherapy]. In joint collaboration of both Institutes the Leipzig-south survey of need of psychotherapy was replicated in a representative sample (N = 1708) of patients waiting one year to be examined and/or treated in out-patient departments of general practitioners and other specialists (gynaecology, orthopaedic surgery and internal medicine) in one district of Prague City (N = 1461) and a rural district in Kromĕríz (N = 247). In a preceding pilot study (N = 207) the zero hypothesis of unspecificity of the Leipzig methodology was rejected. Roughly corresponding proportions of three classes of the need of psychotherapy (defined by means of cluster analysis) were found in the geographic areas, compared in waiting rooms of all out-patient departments except the evidently lower need of psychotherapy found in the rural GP's waiting rooms. Highest rates were those in waiting rooms of gynaecologists and orthopedic surgeons. The GHQ-60 and the Leipzig classifications were compared in both CS samples: 35.1% of male and 30.6% of female respondents did not need psychotherapy and very probably were not psychiatric cases. In 32.5% of males and 36.9% of females the need of generally oriented psychotherapy was combined with suspected hidden psychopathology. 3.8% male and 6.9% female respondents were very probably hidden psychiatric cases and in need of specifically oriented psychotherapy. Predictors of both measures of hidden psychopathology used were obtained from descriptions of personality, demographic background and health care need and attitudes case history by means of multivariate statistical techniques. 12 of 23 descriptors explained 48.8% variance of the global GHQ-60 score. An agreement of 62.9% of the need of psychotherapy back classification was attained by discriminant function using 11 of 23 descriptors. In both procedures the factor analysis derived dimensions of personality were found to be the most potent class of predictors.


[Epilepsy and intelligence. I. Intellectual status in a group of 215 ambulatory patients]. A group of 119 men and 96 women, mean age 32.8 years, with a mean onset of the disease at the age 13 years was examined by Wechsler's intelligence test for adults (Wechsler-Bellevue). They assessed the mean intellectual level, total IQ mean = 104.4, s = 15.3. The intellectual performance of female patients had roughly a normal distribution, in men it was shifted slightly to higher zones. The mean absolute difference of the verbal IQ and performance IQ was mean = 8.4, s = 7.0, median = 7. The results are consistent with some more recent data in the literature on good intellectual abilities of the majority of epileptic subjects. The authors discuss the causes of frequent erroneous ideas of laymen and health professionals on an association of epilepsy and intellectual inadequacy. Among others there persists prejudice, problems of patients as regards memory, problems in the psychosocial sphere, psychopathological manifestations, adverse side-effects of drugs, sequelae of transient impairment of consciousness manifested clinically by short-term reduced performance. Real impairment of epileptic patients are assessed best by special neuropsychological methods, however, evaluation of intellectual abilities is necessary.


[Structural changes in the brain in schizophrenia and affective psychoses]. Kraepelin (1896) and Bleuler (1916) were convinced that schizophrenia has an anatomical basis. Evidence is provided now by visualization methods made in vivo and stereometric methods used in neuropathology. Schizophrenia is associated with enlargement of the lateral and the third ventricle, widening of the sulci between gyri of the cerebral cortex, a decline of the weight of the brain, volume of the cerebral cortex and central gray matter. Histological examination reveals reduction of the parahippocampal gyrus thickness, changes in the hippocampus are controversial. Numerical atrophy of neurons was found in the mediodorsal nucleus of the thalamus, amygdaloid nucleus, prefrontal cortex, impaired pattern of neuronal modules were found in the g.cinguli. Some anatomical changes correlate with differences revealed by neuropsychiatric examination, positron emission tomography and examination of evoked potentials. Schizophrenia can be at least in some instances the consequence of developmental disorder of the brain. The problem of continuity of schizophrenia and affective psychoses is discussed.


Syntheses of cerulenin and its analogs. II. Synthesis and biological activity of dl-carbacerulenin, a carbocyclic analog of cerulenin. 2,3-Epoxy-4-hydroxy-4-((E,E)-3,6-octadienyl)cyclopentanone (dl-carbacerulenin 5) was synthesized via the epoxyketones 15a and 15b as a mimic of the active form of the antibiotics cerulenin 1, a potent inhibitor of fatty acid synthetase (FAS). The monobenzyl ethers (12 and 13), synthetic intermediates of 15, were prepared by direct benzylation of the epoxycyclopentene (7). Inhibitory activity of synthesized 5 toward yeast FAS was less than that of cerulenin by a factor of 1000.


Isoflavanones from the heartwood of Swartzia polyphylla and their antibacterial activity against cariogenic bacteria. The methanolic extract of Swartzia polyphylla DC. heartwood had antibacterial activity against cariogenic bacteria, the mutans Streptococci. The chromatographic purification of the extract afforded seven flavonoids. Among them, three known isoflavanones, dihydrobiochanin A, ferreirin and darbergioidin, and one new isoflavanone, 5,2',4'-trihydroxy-7-methoxyisoflavanone (dihydrocajanin) had potent antibacterial activity against cariogenic bacteria. This effect was not detected on isoflavone derivatives. A comparative antibacterial study of various flavonoids was further performed, and their structure-activity relationship was discussed.


Structures and conformations of metabolites of antitumor cyclic hexapeptides, RA-VII and RA-X. Metabolites of antitumor cyclic hexapeptides, RA-VII and -X which were isolated from Rubia cordifolia were studied by hepatic microsomal biotransformation in rats and in bile juice of rabbits to which these drugs were administered intravascularly. Their structures and conformations were elucidated by two-dimensional nuclear magnetic resonance techniques, temperature effect on NH protons and nuclear Overhauser effect experiments. Specific N-demethylation of Tyr-3, O-demethylation and hydroxylation at aromatic rings of Tyr-3 and -5 were observed. Compared with metabolites of RA-VII, most of RA-X was excreted unchanged in the bile juice. Relationship among their structures, conformations and antitumor activities is also discussed.


Three new oleanene glycosides from Sophora flavescens. Three new oleanene glycosides, sophoraflavosides II-IV (2-4) were isolated together with sophoraflavoside I (1) as the corresponding methyl ester forms from Sophorae Radix, the fresh roots of Sophora flavescens AITON (Leguminosae). Their structures have been elucidated as oxytrogenin 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-beta -D- glucuronopyranoside (2), 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-beta -D-glucuronopyranosyl oxytrogenin 22-O-alpha-L-arabinopyranoside (3) and 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-beta -D-glucuronopyranosyl oxytrogenin 22-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranoside (4), along with unambiguous characterization as 3 beta,22 beta,24-trihydroxyolean-12-en-29-oic acid for their sapogenol, named oxytrogenin (5) on the bases of chemical reactions and spectral analyses.


Triterpene glycosides from the bark of Robinia pseudo-acacia L. I. From the bark of Robinia pseudo-acacia L., five new triterpene glycosides, robiniosides A-D (3, 5-7) and compound III (4), were isolated and their structures were elucidated as 3-O-alpha-L-rhamnopyranosyl(1-->2)-beta-D-glucopyranosyl(1-->2)-beta-D- glucuronopyranosyl 3 beta,22 beta-dihydroxyolean-12-en-29-oic acid (3), 3-O-alpha-L-rhamnopyranosyl(1-->2)-beta-D-galactopyranosyl(1-->2)-beta-D -glucuronopyranosyl 3 beta,22 beta,24-trihydroxyolean-12-en-29-oic acid (4), whose sapogenol was unambiguously characterized and designated as oxytrogenin, 3-O-alpha-L-rhamnopyranosyl(1-->2)-beta-D-glucopyranosyl(1-->2)-beta-D glucuronopyranosyl oxytrogen (5), 3-O-alpha-L-rhamnopyranosyl(1-->2)-beta-D galactopyranosyl(1-->2)-beta-D-glucuronopyranosyl oxytrogenin 22-O-alpha-L-rhamnopyranoside (6), 3-O-alpha-L- rhamnopyranosyl(1-->2)-beta-D-glucopyranosyl(1-->2)-beta-D-glucuronop yranosyl oxytrogenin 22-O-alpha-L-rhamnopyranoside (7), respectively, together with two known triterpene glycosides, kaikasaponin III (1) and 3-O-alpha-L-rhamnopyranosyl(1-->2)-beta-D-galactopyranosyl(1-->2)-beta-D - glucuronopyranosyl 3 beta,22 beta-dihydroxyolean-12-en-29-oic acid (2).


High performance liquid chromatographic determination of bound sulfide and sulfite and thiosulfate at their low levels in human serum by pre-column fluorescence derivatization with monobromobimane. A sensitive and specific high performance liquid chromatographic method for the determination of sulfide, sulfite, and thiosulfate was established. Inorganic sulfur anions were converted into fluorescent derivatives with monobromobimane. The derivatives were separated on a coupled column chromatography with a reversed-phase octadecyl silica column connected with a weakly basic anion exchanger column by isocratic elution with acetic acid solution (pH 3)-acetonitrile (13:3, v/v) containing 25 mM NaClO4. The method was applied to the determination of bound sulfide and sulfite and thiosulfate in normal human serum. Thiosulfate could be determined directly by use of an ultrafiltered sample. For the determination of bound sulfide and sulfite, the pretreatment step with continuous flow gas dialysis was effective for the sample after releasing sulfide and sulfite by reduction with dithiothreitol. The limits of quantification by the present method were 0.05 microM for thiosulfate, 0.5 microM for bound sulfide, and 0.2 microM for bound sulfite.


Studies on thermophile products. V. Immunosuppressive profile in vitro of Bacillus stearothermophilus component, Fr.5-B. The immunosuppressive profile of Bacillus stearothermophilus UK563 component, Fr.5-B, is presented in in vitro studies. Fr.5-B (0.1-1000 ng/ml), provided it was added at the initiation of mixed leukocyte reaction (MLR), inhibited dose-dependently the incorporation of tritiated thymidine ([3H]TdR) into mouse spleen cells and human peripheral blood lymphocytes. Even the addition of Fr.5-B 48 h after the onset of culture suppressed mouse MLR, unlike cyclosporin A (CYA). Fr.5-B significantly inhibited cytotoxic T lymphocyte generation determined by [3H]TdR-release micro-cytotoxicity assay by using mouse mastocytoma P815 as targets. Moreover, this component decreased dose-dependently the expression of class II major histocompatibility molecules (Ia) on mouse peritoneal macrophages induced by concanavalin A supernatant. The present results revealed the unique immunosuppressive property of Fr.5-B which was different from that of CYA.


Inhibitory effects of glycyrrhetic acid derivatives on 11 beta- and 3 alpha-hydroxysteroid dehydrogenases of rat liver. Glycyrrhetic acid (GA), an aglycone of glycyrrhizin (GL), is a potent inhibitor of 11 beta- and 3 alpha-hydroxysteroid dehydrogenases. 11 beta-Hydroxysteroid dehydrogenase activity of rat liver microsomes was potently inhibited by GA, 3-deoxyglycyrrhetic acid (3-deoxyGA), 3-ketoglycyrrhetic acid (3-ketoGA), 3-epiglycyrrhetic acid (3-epiGA) and 11-deoxoglycyrrhetic acid (11-deoxoGA), with I50 values of 2-4 x 10(-7) M. However, 18 alpha-stereoisomers (I50 = 3-7 x 10(-6) M) of GA, 3-deoxyGA and 11-deoxoGA were one tenth less inhibitory on the enzyme activity than the corresponding 18 beta-isomers. On the other hand, 18 alpha-stereoisomers of GA, 3-deoxyGA and 11-deoxoGA inhibited 3 alpha-hydroxysteroid dehydrogenase activity of rat liver cytosol more potently than the corresponding 18 beta-isomers. I50 values of 18 alpha- and 18 beta-isomers were 2 and 7 x 10(-6) M, respectively, in the case of GA, 8 and 20 x 10(-6) M in 3-deoxyGA, 3 and 20 x 10(-6) M in 11-deoxoGA. These results indicate that the 18 beta-conformation of oleanane is important for the inhibition of 11 beta-hydroxysteroid dehydrogenase but on the contrary the 18 alpha-conformation is important for the inhibition of 3 alpha-hydroxysteroid dehydrogenase.


An acidic polysaccharide having immunological activities from the rhizome of Cnidium officinale. An acidic polysaccharide, designated as cnidirhan AG, was isolated from the rhizomes of Cnidium officinale Makino. It was homogeneous on electrophoresis and gel chromatography, and its molecular mass was estimated to be 5.1 x 10(4). It showed pronounced reticuloendothelial system-potentiating activity in a carbon clearance test, and had a remarkable effect on both anti-complementary and alkaline phosphatase-inducing activities. It is composed of L-arabinose: D-galactose: D-glucuronic acid in the molar ratio of 2:6:1, in addition to small amounts of O-acetyl groups. Methylation analysis, carbon-13 nuclear magnetic resonance, controlled Smith degradation and limited acid hydrolysis indicated that the core structural features of cnidirhan AG include a backbone chain composed of beta-1,3-linked D-galactose residues. Some of the galactose units in the backbone carry beta-D-galactosyl side chains at position 6. Both alpha-L-arabinosyl arabinose side chains and terminal beta-D-glucuronic acid residues are linked to the core galactan units.


Design and preparation of pulsatile release tablet as a new oral drug delivery system. To achieve time-controlled or site specific delivery of a drug in the gastrointestinal tract, an orally applicable pulsatile drug release system with the dry-coated tablet form was developed. The system consisted of a less water permeable outer shell and a swellable core tablet; from such a system, the drug was expected to be rapidly released after a certain period of time on the basis of time-controlled disintegration mechanism. Various model disks of outer shell, consisting of hydrogenated castor oil and polyethyleneglycol 6000, were tested for their water penetration rate. The experimental results showed that water penetration proceeded obeying the boundary retreating mechanism, so that the lag time of the system could be controlled by changing either the thickness or the composition of the outer shell. The swelling force of various commercially available disintegrants was quantitatively compared, and it was found that carboxymethylcellulose calcium was the preferable disintegrant to be used for the core tablet. On the basis of the results of a series of fundamental studies, various pulsatile release tablets of isoniazide with different lag times were designed. In the in vitro dissolution test, typical pulsatile release was achieved for all the tablets prepared, and a good correlation was found between the observed lag time and the estimated lag time calculated from an empirical equation deduced from the thickness and polyethyleneglycol 6000 content of the outer shell.


Absorption enhancement of polypeptide drugs by cyclodextrins. I. Enhanced rectal absorption of insulin from hollow-type suppositories containing insulin and cyclodextrins in rabbits. The absorption of insulin (from porcine pancreas) from the rectum of rabbits after the administration of hollow-type suppositories containing insulin and five kinds of cyclodextrins (CyDs) was investigated. Three types of suppositories were employed: suppository I containing insulin (approximately 26 IU/mg) and various amounts of each CyD in citric buffer solution at pH 3.0 or powder in its cavity, suppository II containing CyD without insulin, and suppository III containing insulin without CyD. Without CyD, the insulin and glucose levels in plasma were unchanged, whereas a significant increase in the plasma insulin concentration and a marked decrease in the glucose levels were found following simultaneous administration of insulin and CyDs by suppository I. The enhancing effect of CyD on rectal insulin absorption (absorption-enhancing effect) by chemically modified CyDs (heptakis(2,6-di-O-methyl)-beta-CyD (DM-beta-CyD) and 2-hydroxypropyl-beta-CyD (HP-beta-CyD)) was higher than those by natural CyDs (alpha-, beta-, and gamma-CyD). The area under the plasma concentration-time curve (AUC) and Cmax of insulin significantly decreased with the preadministration (administration of CyD 6, 24 and 48 h before rectal insulin administration) of DM-beta-CyD. The absorption-enhancing effect disappeared 24 h after preadministration. These results suggest that CyDs enhance insulin absorption from the rectum, and that attenuation of the membrane transport barrier function in the rectum recovers at a maximum of 24 h after administration of CyDs.


Photo-stability of the new antiplatelet agent, KBT-3022 (ethyl 2-[4,5-bis(4-methoxyphenyl)thiazole-2-yl]pyrrol-1-ylacetate) in aqueous solutions containing acetonitrile. The photo-stability of ethyl 2-[4,5-bis(4-methoxyphenyl)thiazole-2-yl]pyrrol-1-ylacetate, KBT-3022 in aqueous solutions containing acetonitrile was investigated under the light of a high pressure mercury lamp. Its main photo-degradation product was assumed to be ethyl 5-hydroxy-5-[4,5-bis(4-methoxyphenyl)thiazole-2-yl]-2-oxo-3-pyrrolin+ ++-1-ylacetate. KBT-3022 was also found to undergo ester hydrolysis by heat in both acidic and basic aqueous solutions, but its hydrolysis was confirmed to be negligible in the range of pH 3-9 at room temperature (25 degrees C). Further, its photo-stability with exposure to the high pressure mercury lamp was comparable to that with exposure to a fluorescent lamp. Therefore, it is considered feasible to simulate the photo-stability of KBT-3022 in aqueous solutions containing acetonitrile by exposure to all other light sources including diffuse daylight, if the cumulative number of photons of the light can be determined by actinometry.


A study of the phase transition behaviours of cholesterol and saturated egg lecithin, and their interaction by differential scanning calorimetry. The thermal behaviours of hydrogenated egg lecithin (PC) and cholesterol (C) in anhydrous form and in aqueous dispersions were studied by differential scanning calorimetry. The role of C in phase transitions of C-PC mixtures prepared by physical mixing or coprecipitating from chloroform has been examined. C underwent a phase transition at 34 degrees C and a second one at 76-78 degrees C which depended on the thermal history of the samples. C lowered the gel to liquid crystalline phase transition temperature (Tt) of the aqueous dispersions prepared from C-PC coprecipitates, broadened the endothermic peak at the Tt, and at 1:1 molar ratio the C-PC system did not undergo the phase transition. The C-PC interaction was observed below the Tt. In aqueous dispersions prepared from C-PC physical mixtures, C did not influence the phase transition and acted like an inert diluent. Pellets prepared from C-PC mixtures formed myelin bodies as a result of their erosion in 0.185 M borate buffer (pH 7.4) at below Tt, but the thermal behaviours of these myelin bodies were different for pellets prepared differently. Addition of bovine serum albumin (BSA) (10%, w/w) to the physical mixtures or coprecipitates of C-PC, or to PC alone did not show any effect on the thermal behaviours of their aqueous dispersions at the Tt.


Relationships between biological potency and electronic states of polychlorinated dibenzofurans and polychlorinated biphenyls. It was found that the differences between the frontier molecular orbital energies (epsilon homo - epsilon lumo = delta epsilon) in polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) have a correlationship with the magnitude of the biological activity which is influenced by both the number and position of chlorine atom substituents on PCDFs and PCBs skeletons. Moreover, it was found that the delta epsilon's values of PCBs are classified into two types which coincide with the well-known classification of PCBs to types of 3-methylcholanthrene and phenobarbital according to their biological activities. The relationship between delta epsilon and biological activity in these xenobiotics suggests that the congeners having small delta epsilon values as 2,3,4,7,8-pentaCDF, 2,3,4,6,7-pentaCDF, 3,4,5,3',4'-pentaCB, and 3,4,5,3',4',5'-hexaCB form stable molecular complexes with an Ah-receptor, e.g. (2,3,4,7,8-PentaCDF-Ah-receptor), while the congeners having large delta epsilon values are strongly suggested to be unstable in a complex formation. Thus, this work presents an explanatory method to help understand the structure-activity relationship of the xenobiotics PCDFs and PCBs.


Studies on the constituents of edible and medicinal plants. III. Effects of seven limonoids on the sleeping time induced in mice by anesthetics. Effects of seven limonoids, obakunone (1), 7 alpha-obakunol (2), 7 beta-obakunol (3), limonin (4), 7 alpha-limonol (5), 7 beta-limonol (6) and nomilin (7), on the sleeping time induced in mice by anesthetics were assayed. All the limonoids, except for 2, shortened the sleeping time induced by alpha-chloralose and urethane. 7 gave the highest reduction rate of sleeping time, and the order of the reduction rate of sleeping time was as follows; 7 > 1 and 3 > 4, 5 and 6. As the chemical structures of these compounds are similar to each other, the relationship between the structure and the effects of limonoids on sleeping time was discussed.


Calculation of skin permeability coefficient for ionized and unionized species of indomethacin. The contribution of ionized and unionized species to the overall permeation of weak electrolytes through the skin was investigated to determine the effect of pH in the vehicle on the permeability of indomethacin (IDM), as a model drug, through hairless rat skin. The permeability of IDM through polydimethylsiloxane (silicone) and poly(2-hydroxyethyl methacrylate) (pHEMA) membranes which may reflect lipid and aqueous pathway, respectively, was also measured for comparison. As the pH in the vehicle increased, there was an exponential increase in the skin permeation rate of IDM. The permeation rate of IDM through the silicone membrane was constant independent of pH, whereas that through the pHEMA membrane increased with increasing pH, similar to the skin permeation. The permeability coefficients of ionized and unionized species through the skin estimated using the skin permeation rates and solubilities of IDM at various pHs were 1.50 x 10(-7) and 2.79 x 10(-5) cm/s, respectively. These results indicated that the permeation of ionized species greatly contributed to the total permeation of IDM at higher pH, and that the total permeation rate of IDM was determined by the permeation of unionized species at lower pH. These contributions depend on the pH and pKa values and the ratio of permeability coefficient of each species. It was also confirmed that the skin has at least two kinds of permeation pathways and these two species permeate through a different pathway.


Absorption of diltiazem in beagle dog from pulsatile release tablet. An orally applicable pulsatile drug delivery system in dry-coated tablet form was prepared using diltiazem hydrochloride as the model drug, and a polyvinyl chloride-hydrogenated castor oil-polyethyleneglycol mixture as the outer shell of the tablet. In vitro drug release from the prepared tablet exhibited a typical pulsatile pattern with a 7 h lag phase (non-drug release period). This dosage form was orally administered to three beagle dogs under non-fasting and fasting conditions, and the plasma concentration level of diltiazem was determined according to time after administration. The result of the in vivo study in non-fasting dogs suggested that the drug could be released in the gastrointestinal tract as in the in vitro test. However, under the fasting condition, a large difference in the plasma concentration profile was found, suggesting that the disintegration time of the tablet tended to be influenced by the feeding condition of subject.


Effect of l-menthol on the permeation of indomethacin, mannitol and cortisone through excised hairless mouse skin. The effect of l-menthol on the skin permeability of mannitol, cortisone or indomethacin was examined by an in vitro penetration technique with hairless mouse skin. The donor solution was prepared with phosphate buffered saline, ethanol:buffered saline (20:80, v/v) or ethanol:buffered saline (20:80, v/v) containing 1% (w/v) l-menthol. Although ethanol showed little enhancing effect, l-menthol in an aqueous ethanol vehicle at pH 7.4 increased the permeability coefficients of mannitol and indomethacin by about 100 times that of the control (an aqueous vehicle) and increased that of cortisone by about 10 times. l-Menthol, however, scarcely enhanced the penetration of indomethacin at pH 3.0, the majority of the species being in unionized form. These results suggested that the menthol-ethanol-aqueous system enhanced skin permeability through a direct effect on the polar and/or lipid pathways, while the thermodynamic activity of the penetrant molecule in the delivery vehicle might also influence the effectiveness of the penetration enhancer.


Enhancing effect of cyclodextrins on nasal absorption of insulin and its duration in rabbits. The absorption of insulin (from porcine pancreas) in rabbits after the nasal administration of aqueous preparations containing insulin and five kinds of cyclodextrins (CyDs) in phosphate buffer solution at pH 7.0 was investigated. Without CyD, the insulin and glucose levels in plasma were unchanged, whereas a marked increase in the plasma levels of insulin and a decrease in glucose concentrations were observed following the simultaneous administration of insulin and CyD such as alpha- and heptakis (2,6-di-O-methyl)-beta-CyD (DM-beta-CyD). The largest enhancing effect on the nasal absorption of insulin was obtained by DM-beta-CyD. To evaluate the duration of the absorption-enhancing effect of CyDs, preadministration (administration of CyD 0.5, 6, 12 and 24 h before insulin administration) was performed. The area under plasma concentration-time curve (AUC) and Cmax of insulin significantly decreased with the preadministration of DM-beta-CyD 6, 12 and 24 h before nasal administration. The absorption-enhancing effect disappeared 24h after the preadministration. These findings demonstrate that CyDs enhance the nasal absorption of insulin, and the recovery of the membrane transport barrier function in nasal mucosa is achieved, at the latest, 24 h after the administration of CyDs.


Effect of d-limonene on the amounts of ethanol and indomethacin penetrated from aqueous gel ointments to rat skin. The amounts of d-limonene, ethanol and indomethacin (IMC) which were transferred from aqueous gel ointments to the skin were determined in rats. The concentration of IMC in the skin correlated well with the plasma concentration of IMC percutaneously absorbed from the gel ointment. The increase of d-limonene concentration in the gel ointments was directly proportional to the accumulation of ethanol in the skin. The amount of ethanol in the skin was closely associated with the percutaneous absorption of IMC. As a possible mechanism for enhancement action of d-limonene and ethanol, it was considered that, at first, d-limonene penetrates into the skin under coexistence with ethanol and may change the barrier structure of the stratum corneum. The transfer of ethanol to the skin is thereby enhanced under the coexistence of d-limonene in the skin. Thus, the permeation of IMC can be promoted due to its affinity with ethanol.


Interaction between polyethylene films and bromhexine HCl in solid dosage form. V. Effect of packaging materials on the sorption of bromhexine HCl. A prevention method of the sorption of bromhexine HCl to plastic materials used in packaging was investigated. Four kinds of plastic packaging materials were used: Polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP) and polyacrylonitrile (PAN). Three polyethylenes having different densities were used. No effect of PE density on the sorption of bromhexine HCl from granules was observed. The effects of different kinds of plastics on the sorption of bromhexine HCl from solution and granules were studied. The sorption of bromhexine HCl to PAN, which had a high relative dielectric constant, was the most depressed among the four plastics. The sorption of meclizine HCl to PAN from the solution was also lowest, the same as bromhexine HCl.


Effects of phenylalaninol on centrally induced gastric acid secretion. The effects of phenylalaninol (D-isomer) on gastric acid secretion and gastric ulcer were studied in rats. The compound reduced the gastric acid secretion stimulated by intracisternal thyrotropin releasing hormone and intravenous 2-deoxy-D-glucose, but not that stimulated by subcutaneous carbachol or histamine. Phenylalaninol prevented stress- and indomethacin-induced gastric ulcers. We conclude that phenylalaninol inhibits ulcer formation mainly by central inhibition of gastric acid secretion.


Thunberginols A, B, and F, new antiallergic and antimicrobial principles from hydrangeae dulcis folium. Six new antiallergic and antimicrobial principles, thunberginols A, B, C, D, E, and F, were isolated from Hydrangeae Dulcis Folium, the fermented and dried leaves of Hydrangea macrophylla SERINGE var. thunbergii MAKINO. The chemical structures of thunberginols A, B, and F have been determined on the basis of chemical and physiocochemical evidence. Thunberginols A, B, and F showed more potent antiallergic activity than phyllodulcin, hydrangenol, and AA-861 in the in vitro test using the Schults-Dale reaction in sensitized guinea pig bronchial muscle. Thunberginols A, B, and F also exhibited antimicrobial activity against oral bacteria.


[Pathological and clinical study of the primary B and T cell lymphomas of intestines]. Thirty-one cases of primary non-Hodgkin's lymphoma of intestine were typed with monoclonal antibodies UCHL1, Ki-B5, L26 and Ki-MiP. Among them, there were 21 B cell lymphomas and 10 T cell lymphomas. Morphologically, the low-grade MALT lymphomas consisted of centrocyte-like cells, accompanying frequently with lymphoepithelial lesions and residual germinal centers, whereas the T cell lymphomas consisted of pleomorphic lymphoma cells, always accompanying with vascular infiltration, massive necrosis, atrophy of intestinal villi, phagocytosis of reactive histiocytes and epithelitropism. Clinically, T cell lymphomas of intestine occur more frequently in the young adults, usually with fever and diarrhea and is worse in prognosis than that of B cell lymphomas of the similar location.


[Histological localization of mucin core polypeptide MUC-2 in application to the differential diagnosis of adenocarcinoma]. Human mucin core cDNA-MUC-2 has been cloned and sequenced. Its major portion consists of amino acids tandem in arrangement with repetition in its sequence, locating at the 11th chromosome. Antibody against polypeptide MUC-2 was used for immunohistochemical SABC staining in order to investigate the expression of MUC-2 in human normal and cancerous tissues. The results revealed that MUC-2 was expressed in normal intestine and prostate, and accumulated in the colorectal cancer tissues, but not in other normal tissues or only weakly expressed in a few cases of pancreas, lung and breast cancers. If used in combining with monoclonal antibody against MUC-1, which is known to be expressed in normal and cancer tissues of pancreas, lung and breast, it is considered to be valuable for the differential diagnosis in detecting the origin of metastatic adenocarcinoma.


[Electron microscopy microanalysis and quantitative detection of trace elements in carcinoma of the colon]. The spectrum of trace elements was determined in 19 cases of colon carcinoma with analytical electron microscope. Besides, the quantities of trace elements in the cancerous tissue and the mucosa beyond the site of cancer were measured in 31 cases by flameless atomic absorption spectrophotometry. A reprocessing technique was applied to convert the paraffin sections directly to resin sections in order to recover the assessment of elements in these tissues. Fe, Al and macro elements Ca, S were recovered in 100% of the cases in both the cancer tissue and the mucosa taken beyond the site of cancer. The recovery rates of Ni and macro elements including K, Mg, P, Cl were 50%. The recovery rates of Si in poorly-differentiated adenocarcinoma were higher than that in the mucosa far from the cancer site. The levels of Cu, Zn and Ni in the cancerous tissue were lower than those detected in the mucosa (P < 0.01). No significant difference was noticed between the cancerous tissue and the mucosa beyond the site of cancer on Fe, Mn, Pb, Co and Cr contents.


[An immunohistochemical and ultrastructural survey on smooth muscle tumors of the alimentary tract]. 33 cases of smooth muscle tumors of the alimentary tract were analysed. Among them, 18 cases showed positive reaction to desmin, including one out of two cases of esophageal neoplasms, 6 out of 11 cases of gastric neoplasms, 5 from 12 cases of small intestinal neoplasms, 6 from 8 cases of large intestinal neoplasms, 14 out of 21 cases of benign tumors as well as sarcomas in grade I, and 4 from 12 cases of sarcomas in grade II and III (only mild positive result obtained in grade II and III-sarcomas). S-100 protein staining was performed in 27 cases and positive reaction was obtained only in five. Among them, 2 cases showed prominent positive reaction. 20 cases were studied by electron microscopy, and cytoplasmic myofilaments as well as dense bodies were demonstrated in 7 cases. All of them were the benign neoplasm or sarcoma in grade I. The conclusion is that most stromal tumors of the digestive system are originating from the smooth muscle, except very few cases are neurogenic in origin.


[Oncogene expression and point mutation in human pancreatic adenocarcinomas]. By nucleic acid hybridization and PCR techniques, oncogene expression in normal human pancreatic tissue as well as 4 human pancreatic adenocarcinoma cell lines were studied. Additionally, c-Ki-ras gene point mutation were also detected in the paraffin embedded sections collected from 34 cases of surgical specimens resected due to pancreatic adenocarcinoma. The results showed that none of the 5 oncogenes described was expressed in the normal human pancreatic tissue, whereas, all the human pancreatic adenocarcinoma cell lines expressed c-myc and c-Ki-ras oncogenes. 28 out of 34 cases (82.4%) of human pancreatic adenocarcinomas had c-Ki-ras gene codon 12 point mutation.


[Phenotypic changes of mast cells during hepatocarcinogenesis in rats]. Sequential observation was made to detect the phenotypic change of mast cells during the process of dimethylaminoazobenzene-induced hepatocarcinogenesis in rats by cytochemistry, immunocytochemistry and electron microscopy. The number of mast cells in the liver tissues increased in various extent 4 weeks following the exposure to carcinogen. The increasing tendency was speeded up as time proceeded. At the 16th week, the number of mast cells reached to seven times as that in the normal rats. The mast cells appeared in the early stage of carcinogenesis were connective tissue mast cells (CTMC). From the 16th week on, mucosal mast cell (MMC) could be detected in some animals, but its number was much less than that of CTMC. In the late stages of experiment, the number of mast cells increased markedly, which showed positive reaction by ARMCP I and ARMCP II staining respectively and were to be brown or blue in color by combined Alcian blue. PAS. ABC staining. At the same time, an inter mediate type of mast cell between MMC and CTMC, light brown in color was also observed. Immunocytochemically, these cells presented ARMCP II positive reactivity. In various kinds of liver tumours formed at 26th week, mast cells occurred in a quite varying amount in different animals. No relationship was found between the amount of mast cells and the cell type or differentiation of the tumours, while the phenotypic distribution of mast cells was closely correlated with the histological type. The development and ultrastructural characterization of mast cells in the portal tracts and sinusoids were also investigated.


[A study of duck hepatitis B virus infection in Chinese ducklings]. Intraperitoneal inoculation of duck hepatitis B virus in three different dosages (9 x 10(7), 1.8 x 10(8), 9 x 10(8) DHBV particles) into 3 to 21 day-old Chinese ducklings provided from a DHBV free flock produced a persistent infection up to 93.3% in 60 animals. The serum and liver specimens of these ducklings were examined by DNA dot blot hybridization on the 30th day after inoculation. The results showed that: (1) examination of viral DNA in liver was more sensitive and reliable than estimation of the DNA in serum for detecting DHBV infection in inoculated ducklings; (2) the liver DHBV DNA level did not coincide with the degree of liver hepatitis induced; (3) 21-day-old Chinese ducklings were also susceptible to DHBV infection, the infection rate of this group was 100% (10/10).


[Study of collagen abnormality in Marfan's syndrome]. Unique picrosirius-polarization method was used to detect the distribution of collagen fibers in aorta and skin of patients with Marfan's syndrome (MS). The adventitia of aortae of MS patients was noticed to be composing of a certain amount of thin type III collagen fibers, and the reticular layer of skin consisted of large amount of thin type III collagen fibers. The results indicated that the distribution of collagen type in Marfan's syndrome becomes abnormal. Hydroxyproline assay analysis showed no significant difference obtained on the total collagen content of aorta and skin between the MS patients and the controls. Anyhow, content of acid soluble collagen in the aortae of MS patients was learnt to be increased significantly (P < 0.01). CNBr-cleavage electrophoresis showed also change of fragment CB8 band in one case.


[Analysis of DNA ploidy in 3,050 cases of malignant tumors]. DNA ploidy in paraffin-embedded sections collected from 3,050 cases of malignant tumors was analysed by flow cytometry. The result showed that DNA index distribution was ranged from 0.6 to 2.6, mostly at 1.10 to 1.85. DNA ploidy distribution showed that 536 cases (17.6%) were DNA diploid tumors; 2,227 cases (73.0%) were tumors with DNA aneuploid; 126 cases (4.1%) accompanied with tetraploid; and 161 cases (5.3%) with multiploid. Additionally, relationship between the histologic type as well as the grading and DNA ploid were also analysed in 1,117 cases of malignant tumors. The results indicated that DNA index was not correlated with the histologic typing, but correlated with the histologic grading (grade I DI = 1.24, grade II DI = 1.38, grade III DI = 1.51). It's considered that flow cytometry DNA grade index may be an objective and precise quantitative marker in judging the degree, grade and differentiation of tumor malignancy.


[Study of the cultured human endothelial cells infected by epidemic hemorrhagic fever virus]. Virus antigen could be detected in the cytoplasm of infected human endothelial cells (HEC) by immunofluorescent assay (IFA) 2 to 10 days after the inoculation of epidemic hemorrhagic fever virus (EHFV), but no apparent histologic changes could be found by phase contrast light microscopy, as well as no mature virus particles could be detected under the transmission electron microscope. Reinoculation of the freeze-melt supernatant of HEC 8 days after the inoculation of EHFV to EHFV susceptible Vero E-6 cells, viral antigen could be detected in most of these cells and mature EHFV particles or viral inclusion bodies could also be obtained in the cytoplasm under transmission electron microscope. The results show that HEC is a susceptible target cell to EHFV and infection by this virus may not give apparent cytopathogenic effect in HEC.


[Endothelial cell injury induced by linoleic acid hydroperoxide and atherosclerosis]. Effect of lipid peroxidation on the development of atherosclerotic lesions was studied in 10 diet-induced hyper cholesterolemic rabbits. By the end of the 3rd week of hypercholesterolemia, lipid peroxidative injury of the endothelial cells was induced by injecting linoleic acid hydroperoxide into a defined segment of the carotid artery. By the end of the 8th and 10th week light and electron microscopy revealed that atherosclerotic lesions mainly the fibrous plaques developed in certain segments of the artery described. The results indicate that under hypercholesterolemic condition, endothelial peroxidative injury may promote and intensify the process of atherogenesis. Furthermore, endothelial peroxidative injury which is characterised by the presence of plasma membrane blebbing can also be induced by hypercholesterolemia.


[Study on the capillary permeability of myocardium in diabetic rats]. The alteration of myocardial capillary permeability (MCP) in experimental diabetic rat was investigated by using cationic ferritin as a tracer. Diabetes was induced by injection of streptozocin (70mg/kg b.w.ip.). Vehicle injected, age and body weight-matched rats were served as the controls. The myocardial tissue was examined by the end of 2,7, and 9 months or even longer after diabetes induction. Results showed that (1) MCP was significantly increased in diabetic rats in comparing with that of the controls; (2) The increase of permeability became more pronounced with prolongation of the course of diabetes; and (3) the increase of MCP preceded the capillary basal laminar thickening demonstrated by electron microscopy. These findings suggest that increased MCP may play a role in the pathogenesis of basal laminar thickening and myocardial complications in diabetic state.


Cells from the early chick optic nerve generate neurons but not oligodendrocytes in vitro. We have recently described neuronal potentialities in neuroepithelial cells of the embryonic chicken optic nerve (Giess et al., Proc. Natl. Acad. Sci. USA, 87 (1990), 1643-1647). To further investigate the developmental repertoire of optic nerve cells, oligodendroglial development was studied in cultures of optic nerve explanted at various developmental stages. Oligodendrocyte differentiation was analyzed using antibodies directed against galactocerebrosides (Gal-C) and against sulfatides. Optic nerves removed at embryonic days 5 and 6 (E5-E6) never gave rise in culture to differentiated oligodendrocytes, even after 3 weeks in vitro. In contrast, in cultures of optic nerves removed from E7 or older embryos, cells expressing both oligodendrocyte markers were rapidly and invariably observed. Absence of oligodendrocytes before E7 was not due to culture conditions being inadequate to support the differentiation of early precursors along this pathway, since neuroepithelial cells from E2 and E4 trunk neural tube cultivated in the same conditions expressed Gal-C after respectively 16 and 10 days. These results demonstrate that the optic nerve territory is initially devoid of oligodendrocyte potentialities. Whether oligodendrocyte precursors that, around E7, populate the optic nerve are induced by a specific developmental signal occurring at this stage or migrate from outside the optic nerve remains to be determined.


Isolation of IFAPa-400 cDNAs: evidence for a transient cytostructural gene activity common to the precursor cells of the myogenic and the neurogenic cell lineages. Differentiation of neural and muscle cells is characterized by a switch in the expression of the type of intermediate filament protein subunit. In these lineages, vimentin is transiently expressed in the initial stages of development and is gradually replaced by a tissue specific protein. We have identified a giant developmentally regulated antigen (IFAPa-400) which colocalizes with vimentin in the precursor cells of the neurogenic and myogenic lineages of the chick embryo [Chabot and Vincent (1990) Dev. Brain Res. 54, 195-204; Cossette and Vincent (1991) J. Cell Sci. 98, 251-260]. Based on the expression of this protein during neurogenesis and myogenesis, we hypothesize that IFAPa-400 and vimentin define a special intermediate filament network, common to the non-differentiated cells derived from the neuroectoderm and those of the myogenic tissues. We report here the isolation and sequence of partial cDNAs encoding more than 400 amino acids of the carboxy-terminus of this protein. RNA blot analysis and in situ hybridization indicate that IFAPa-400 represents a bona fide developmentally regulated gene product. These results further confirm that IFAPa-400 mRNA transcripts are limited to the early precursor cells of both neurogenic and myogenic lineages.


Testosterone fails to save androgen-sensitive rat motoneurons following early target removal. Axotomy during development can result in the death of up to 100% of the affected motoneurons. However, axotomy-induced death can be significantly reduced by administration of androgens in young rats. Motoneuron death in the spinal nucleus of the bulbocavernosus (SNB) has previously been shown to be regulated by androgens during development. the present experiment examined the effects of androgen treatment on the survival of SNB motoneurons after target removal and concomitant axotomy early in development. On the day of birth, two target muscles of SNB motoneurons of male and female rats were bilaterally extirpated. Target removal resulted in a dramatic loss of SNB motoneurons within 48 h of surgery, with an ultimate loss of virtually all motoneurons projecting to the extirpated muscles by postnatal day 10. Treatment with testosterone failed to save SNB motoneurons from target removal-induced death. Pups treated with testosterone after target removal did not differ in the pattern or timing of motoneuron loss from untreated pups at any age examined. Counts of degenerating cells in the SNB reflected the extensive motoneuron loss and also did not differ with testosterone treatment. These results indicate that testosterone cannot save the androgen-sensitive SNB motoneurons from death after target removal and concomitant axotomy early in development. The failure of testosterone treatment to rescue SNB motoneurons in the absence of the SNB target musculature further suggests that during normal development, both androgens and target muscles are necessary for SNB motoneuron survival.


Plasticity and ontogeny of the central 5-HT transporter: effect of neonatal 5,7-dihydroxytryptamine lesions in the rat. 5,7-Dihydroxytryptamine (5,7-DHT) is unique as a serotonin (5-HT) neurotoxin in that i.p. injection of neonatal rats increases concentrations of 5-HT in brainstem while depleting 5-HT in cortex, hippocampus and spinal cord. To study the mechanism of this effect we measured the 5-HT transporter or uptake site, a presynaptic marker, using [3H]paroxetine binding. There were significant regional differences in Bmax of vehicle-injected rats: brainstem, diencephalon > striatum, cortex, spinal cord > hippocampus, cerebellum. There were also regional differences in the ontogeny of bindings sites: at postnatal day 7, [3H]paroxetine sites were 39% of adult levels in cortex compared to 63% in brainstem. Thirty days after 100 mg/kg 5,7-DHT i.p., Bmax of [3H]paroxetine binding was significantly increased in brainstem (+67%) and diencephalon (+136%), whereas it decreased in cortex (-59%), hippocampus (-94%) and spinal cord (-99%), striatum (-41%) and cerebellum (-37%). KD remained unaltered. In dose-response studies (0-200 mg/kg), 50 mg/kg was the threshold dose for Bmax effects and 200 mg/kg was lethal. In weekly time-course studies, changes were apparent 1 week after 5,7-DHT lesions. Binding site increases in diencephalon and brainstem were not maximal until 3 weeks after injection, whereas percent decreases in cortical sites remained unchanged at each week studied. Lesion effects on the ontogeny of [3H]paroxetine binding sites were region-dependent: cortical sites continued to increase with age but spinal sites did not. There was no significant recovery in spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)


Rat embryonic septal neurons survive and express cholinergic properties in isolation and without nerve growth factor. We studied survival and expression of cholinergic properties in embryonic septal neurons grown in very low density microcultures (1-7 cells per Terasaki well). Even in cultures containing only a single neuron, at least 10% of plated neurons survived for 2 weeks or more in medium containing fetal calf serum or an acid-stable fraction (55,000 Da) of horse serum. Of these surviving neurons, 30-40% stained positively for acetylcholinesterase (AChE) or nerve growth factor (NGF) receptor, even though the culture medium lacked detectable levels of NGF, brain-derived neurotrophic factor, and fibroblast growth factor. Addition of NGF or an antibody against NGF had no effect on either neuronal survival or the percentage of neurons staining positively for AChE or NGF receptor after 18-20 days in vitro. There was no cell division in medium containing the serum fraction, but when 10% fetal calf serum was present cell division occurred in some of the cultures, and in half of these cases at least one of the clonal progeny became AChE-positive. These results demonstrate that some embryonic septal cells can survive at least 2 weeks and develop cholinergic neuronal properties in the absence of other cells or NGF.


Transient pattern of exuberant projections of olfactory axons during development in the rat. The purpose of our study was twofold: (1) to trace the development of the olfactory axons from early embryonic stages until the mature pattern of connectivity and (2) to determine whether a transient penetration of them exists beyond the olfactory glomeruli. Two techniques were employed: DiI applied in the olfactory epithelium after aldehyde fixation, and olfactory marker protein (OMP) immunostaining. At E13 and E14 olfactory axons were observed spreading over the telencephalic vesicle and entering deeply into the prospective olfactory bulb, extending near the ventricular zone. Growth cones were seen at the end of these axons. At E15, the bundles of olfactory axons form a network, in which axons, growth cones and cells were seen. Some of these axons entered the olfactory bulb. Using OMP immunostaining olfactory axons were observed along the external plexiform layer, the mitral cell layer and in the granular layer from E19 to P6. At P9 some OMP immunoreactive axons were observed in the external plexiform layer. No OMP immunostained axons could be observed outside the glomeruli at P10. Our conclusions are that a transient immature pattern of early invasion over the telencephalic vesicle and of the olfactory bulb by olfactory axons occurs in the olfactory system. By the second postnatal week the glomerular layer reaches its mature configuration, and no olfactory fibers are seen outside the glomerular layer.


Ontogeny of hippocampal afterdischarges in the urethane-anesthetized rat. Experimental studies have shown that seizure manifestations vary as the brain develops. This study investigated the characteristics of afterdischarges in the hippocampal circuits at various ages in the developing rat. Rats from the following post-natal periods were tested: PN 10-11, PN 14-15, PN 17-19, PN 21-23 and PN 25-27. Animals were anesthetized with urethane and recording electrodes placed in the hippocampus bilaterally. Stimulating electrodes were placed in the left CA3 region and in the angular bundle. Afterdischarges were produced in all animals using stimulus trains of 20 or 50 Hz. Rats in the PN 10-11 and 14-15 age groups had afterdischarges that consisted of population spikes in CA1 and broad positive potentials in the dentate gyrus. Between PN 17 and 19, maximal dentate activation, which consists of bursts of large amplitude population spikes in the dentate gyrus, first appeared in response to 20 Hz stimulation to CA3 or either 20 or 50 Hz stimulation to the angular bundle. Rats older than 21 days had afterdischarge patterns like those recorded in the adult. These data indicate that, in the rat, the seizure capabilities of the limbic circuits go through a major transition period around PN 17-19. The appearance of maximal dentate activation marks the ability of the developing rat brain to produce and sustain reverberatory seizure discharges.


Ontogeny of a sexually dimorphic nucleus in the preoptic area of the Japanese quail (Coturnix japonica). The nucleus preopticus medianus (POMn) is a sexually dimorphic nucleus in Japanese quail (Coturnix japonica) that is critically involved in the hormonal activation of male copulatory behavior. The larger volume apparent in males appears to depend upon circulating testosterone [Brain Res., 416 (1987) 59-68; J. Comp. Neurol., 303 (1991) 443-456]. The present study determined when during normal development this nucleus becomes dimorphic. POMn and a control nucleus, the nucleus commissurae pallii (nCPa), were traced from Nissl-stained coronal sections (40 microns) from animals sacrificed at 2, 3, 4, 5, 6 or 7 weeks of age. Areas were measured and used to calculate volume. POMn volumes were not significantly different in males and females through 5 weeks of age. The dimorphism in POMn volume then became apparent at 6 weeks of age as a function of an increase in male POMn volume between 5 and 6 weeks of age. No significant differences were apparent at any developmental age in nCPa volume. The appearance of a sexual dimorphism in POMn volume is coincident with the pubertal surge in testosterone that occurs between 5 and 6 weeks of age [Horm. Behav., 11 (1978) 175-182], and is also coincident with behavioral sexual maturity.


Localization and developmental expression of a novel protein kinase C delta gene. The expression and localization of a novel protein kinase C delta (nPKC delta) mRNA were investigated using Northern blotting and in situ hybridization in the developmental process of mouse brain. In adult mice, nPKC delta was abundantly expressed in the thalamus, moderately in the pons and the cerebellum, but faintly in the cerebral cortex and the spinal cord. By in situ hybridization, the signals were observed specifically at the sensory and motor relay nuclei of the thalamus, the dorsal cochlear nuclei of the pons, and the molecular layer of the cerebellum. When developmental changes in the expression of nPKC delta gene were analyzed by in situ hybridization, it was not detectable in embryonic and neonatal brains, very weakly expressed in the thalamus in the first week, and highly expressed at two weeks of age. These results suggest that the gene expression of nPKC delta is strictly controlled by both the cell type and the developmental process.


Pre- and post-natal ontogeny of thyrotropin-releasing-hormone in the rat spinal cord: an immunocytochemical study. This work aimed at providing by means of immunocytochemical techniques a detailed study of the ontogeny of thyrotropin-releasing hormone (TRH) in the spinal cord of the rat. We report the first appearance of TRH-immunoreactive fibers in the ventral funiculus of thoracic and lumbar levels at embryonic day 17. At embryonic day 18, fibers penetrated the ventral gray matter towards the central canal. At embryonic day 19, the first immunoreactive fibers were seen in the intermediolateral cell column at upper thoracic levels. This region was invaded at lower thoracic levels on the day of birth. At this time, TRH-immunoreactive axodendritic synapses were observed in the ventral horn and in the intermediolateral cell column. Immunoreactivity increased in these regions until post-natal day 21 when the adult pattern of TRH immunoreactivity was established in the sympathetic nuclei and in the ventral horn. However, a transient TRH-like immunoreactivity was detected in lamina IIi of the dorsal horn between post-natal days 14 and 30: at ultrastructural level, immunoreactive varicosities were seen to establish axodendritic synapses. In conclusion, TRH is one of the earliest peptidergic systems established in the spinal cord and it presents extensive temporal and topographical similarities with the serotonergic system with which it could be colocalized.


Acetyl-L-carnitine has a neuromodulatory influence on neuronal phenotypes during early embryogenesis in the chick embryo. Studies from this laboratory and others have demonstrated that neuroblasts in early embryogenesis exhibit a high degree of plasticity with respect to neurotransmitter phenotype. The critical period within which these neuroblasts are sensitive to the effects of endogenous neurotrophins has been defined as 1-3 days of development in the chick embryo. In this study, we examined the influence of acetyl-L-carnitine (ALCAR) administered in ovo during embryonic days 1-3 (E1-E3) and sacrificed at embryonic day 8 (E8) on cholinergic and GABAergic neuronal phenotypes using as neuronal markers the activities of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD), respectively. Phenotypic expression was assessed in 3 distinct anatomical regions of the embryonic brain: cerebral hemispheres (CH), optic lobes (OL), and diencephalon-midbrain-brainstem (DMBS). A single administration of ALCAR at embryonic day 1 resulted in a dose-dependent increase in ChAT activity and decrease in GAD activity in CH. ChAT activity was again increased and GAD activity decreased in CH from embryos that were administered ALCAR (100 micrograms/50 microliters/day) daily from embryonic day E1 to E3. No change was observed in either ChAT or GAD activity in OL in response to ALCAR administration during the critical period (E1-E3) at doses ranging from 10 to 500 micrograms/day. In the DMBS, the activity of ChAT exhibited a marked increase at lower doses (10 micrograms) followed by a marked decrease at higher doses (500 micrograms) of ALCAR. The decrease in ChAT activity in DMBS was again observed at an ALCAR dose of 100 micrograms/day when administered from E1 to E3.(ABSTRACT TRUNCATED AT 250 WORDS)


Expression of the HBNF (heparin-binding neurite-promoting factor) gene in the brain of fetal, neonatal and adult rat: an in situ hybridization study. HBNF (heparin-binding neurite-promoting factor) and MK (midkine) are members of a newly recognized family of proteins, the expression of which is developmentally regulated. These proteins are expressed highest during fetal development in many tissues but they seem to be rather restricted to the brain in adult animals. Gene expression for these proteins is inducible by retinoic acid in embryonal carcinoma cell lines. They induce neurite outgrowth in cultured neurons, and they are characterized by high sequence conservation between species. While the function(s) of these proteins are unknown, available evidence suggests possible roles in the development and the maintenance of neural tissues. This in situ hybridization study investigates the temporal and spatial expression pattern of the HBNF gene in the brain of developing rats. The HBNF gene is highly expressed in the neuroepithelium and the ependyma from fetal day 15 on. Although most ependymal structures express the gene strongly, a few restricted areas of the ependyma do not express HBNF (ventral part of the fourth ventricle, subcommissural organ). In the brain parenchyma, HBNF is expressed in the thalamo-hippocampal area from fetal day 15 and in the cerebral cortex from fetal day 16, with high expression occurring in the superficial layers of the cortex. The nature of the cells expressing the gene, while difficult to ascertain, is probably glial for the most part. However, certain neurons (in limited areas of the brain parenchyma) and most pial cells (in the meninges), also express the gene. HBNF gene expression decreases sharply a few days after birth. HBNF mRNA is also detectable at fetal days 15 and 16 in the face fetal mesenchyma. In the adult rat brain, the expression of the HBNF gene appears to be restricted to neurons of the hippocampus and of the olfactory bulb and to the superficial layers of the cortex. The structurally related MK gene, though not extensively studied here, shows an entirely different temporal and spatial expression pattern. MK gene is weakly expressed during ontogeny in most brain areas, and in the adult animal, MK mRNA is present only in the choroid plexus. The intense and widely distributed expression of the HBNF gene in several cell populations in the fetus, the progressive spatial and quantitative restriction of HBNF gene expression with brain differentiation, as well as the properties of the protein suggest important and diverse functions for HBNF in cellular interactions and cell differentiation in the developing brain, that must act temporally and spatially by ways distinct from its MK companion molecule.


The role of olfactory bulb norepinephrine in early olfactory learning. Wistar rat pups were implanted with bilateral olfactory bulb cannulas on postnatal day 5 (PN5). On PN6, pups were trained in an olfactory classical conditioning task with peppermint odor as the CS and tactile stimulation/stroking as the UCS. Pups were randomly assigned to either PAIRED, BACKWARD or ODOR-only conditions. Half the pups in each group received intrabulbar infusions of 100 microM propranolol and half received intrabulbar infusions of saline during the training session. Propranolol infusions blocked acquisition of the learned odor preference expressed by PAIRED saline-infused pups. Diffusion of the infusate was checked in additional pups by infusing [3H]NE and performing LSC analysis. Infusate concentration did not significantly differ between the anterior and posterior halves of the bulb, but were sharply lower in the olfactory peduncle and more posterior areas. The results suggest that olfactory bulb NE is critical for early olfactory learning.


Local perineal implants of anti-androgen block masculinization of the spinal nucleus of the bulbocavernosus. Female rats were injected with testosterone propionate on the 1st and 3rd days of life. In addition, some females received 200 micrograms of the anti-androgen, hydroxyflutamide. Females receiving anti-androgen directly to the perineum, including the muscles bulbocavernosus and levator ani, had fewer surviving spinal nucleus of the bulbocavernosus (SNB) motoneurons than did females receiving the anti-androgen systemically. These results indicate that androgen acts upon the target muscles to spare developing SNB motoneurons from death.


A narrow window of intracellular calcium concentration is optimal for neurite outgrowth in rat sensory neurones. We have examined neurite outgrowth in rat sensory neurones when cytosolic free calcium concentration ([Ca2+]i) was varied in the range 0-60 nM. Neurite outgrowth was maximal at 35 nM [Ca2+]i and was reduced at higher and lower values of [Ca2+]i. These results provide direct evidence for Mattson and Kater's suggestion of an optimal calcium range for growth cone function.


Canadian Society of Clinical Chemists position paper: standardization of selected polypeptide hormone measurements. This Canadian Society of Clinical Chemists (CSCC) sponsored position statement addresses the problem of nonuniformity of standardization of polypeptide hormone analyses. First we review the structural diversity of selected polypeptide hormones including growth hormone (GH), prolactin, follitropin (FSH), lutropin (LH), human chorionic gonadotropin (hCG), and thyrotropin (TSH) and the problem that this heterogeneity creates. The history and availability of reference materials for the analyses of these hormones are then summarized. Finally we make a list of recommendations regarding standardization, methods of measurement, and reporting of polypeptide hormone results. Implementation of the recommendations will lead to improvement in inter-laboratory comparability and more useful hormone assays.


Mechanism of differential inhibition of lactate dehydrogenase isoenzymes in the BMC LD-1 assay. The mechanism of inhibition of lactate dehydrogenase (LD) isoenzymes by guanidinium thiocyanate (GSCN) used in the LD-1 assay developed by Boehringer Mannheim Corporation (BMC) was investigated. Michaelis-Menten inhibition kinetics for the individual isoenzymes revealed that GSCN competitively inhibited LD-1 in the presence of lactate and NAD+, but is a noncompetitive inhibitor of LD-5. LD-2 and LD-3 exhibited mixed inhibition kinetics. The inhibition constants were two- to threefold smaller for LD-5 than for LD-1. Time-dependent studies also showed that the isoenzymes underwent a different rate of inactivation by GSCN. LD-5, LD-3, and LD-2 were rapidly inactivated within 1 min under the BMC assay conditions, whereas LD-1 lost only about 20% of activity after 10 min. The presence of lactate further protects LD-1, but not other isoenzymes. Under this condition, LD-1 was not inactivated during the initial 6 min of reaction. Separate experiments demonstrated that both guanidinium and thiocyanate ions are responsible for the inactivation process that was found to be irreversible. We speculate that GSCN selectively denatures the M subunit of LD. The H subunit is less susceptible to denaturation and is further stabilized by lactate.


A novel colorimetric method for assaying arginase activity. Arginase catalyzes the conversion of arginine to urea and ornithine in the liver of ureotelic animals. Higher activity of this enzyme is found in tumors as well as in the sera of patients with hepatic diseases. We have developed a simple colorimetric method for its determination. This is based on the determination of residual arginine, after its conversion with p-nitrophenyl glyoxal (PNPG) at pH 9.0 in the presence of sodium ascorbate. The reaction product obeys Beer's law in the range of 0.01-0.20 mmol/L arginine with an arginine-equivalent molar extinction coefficient of 0.65 x 10(4) M-1 cm-1. The decrease in absorbance in the presence of arginase correlates with the enzyme activity. Color development as well as termination of enzyme activity is achieved by addition of a single reagent, thereby obviating the use of many chemicals necessary in other methods. The sensitivity of this method is equivalent to those of currently available procedures but has the added advantages of greater convenience.


Heterogeneity of hairy cell tartrate-resistant acid phosphatase. The human nonerythrocytic acid phosphatases (AcP) are composed of seven distinct activity bands in nondenaturing polyacrylamide gel electrophoresis (PAGE) when stained using either 1-naphthyl phosphate or naphthol ASBI phosphate as substrate. They are numbered 0, 1, 2, 3, 3b, 4, and 5 according to their increasing mobility toward the cathode in acidic conditions. Of these, only the most cationic "band 5" is tartrate resistant (TRAcP). When naphthol ASBI phosphate is used as substrate, AcP activity can also be stained in situ. In the presence of tartrate, activity remains strong in the hairy cells (HC) of hairy cell leukemia (HCL). Thus, the TRAcP stain has remained a reliable marker for HC. To investigate the function of TRAcP in HC, we purified two isoforms of TRAcP from HCL spleen tissue and found them to have similar substrate specificities and inhibitor sensitivities. In this report, we describe in detail the methods for TRAcP purification and compare some of the structural properties of the two isoforms to reinforce the concept that human TRAcP is a heterogeneous group of related enzymes. Band 5 represented only 15-20% of the total TRAcP extracted from HCL spleen. The remaining 80% of TRAcP hydrolyzed p-nitrophenyl phosphate but not naphthol ASBI phosphate and was not detectable in acidic, nondenaturing PAGE gels. Band 5 was solubilized from tissue using 500 mmol/L NaCl after previous extraction with 0.5% (v/v) NP-40 removed most other AcP and TRAcP activity.(ABSTRACT TRUNCATED AT 250 WORDS)


Antibodies to the p53 tumor suppressor gene product quantified in cancer patient serum with a time-resolved immunofluorometric technique. We have developed new methodology for quantifying antibodies to the p53 tumor suppressor gene product in human serum. The assay involves solid-phase immobilization of a monoclonal anti-p53-specific antibody that is then reacted with a tumor cell line lysate containing mutant p53. The immunopurified p53 antigen acts as an immunosorbent for the serum p53 antibodies that are then detected by reaction with a goat anti-human immunoglobulin G antibody labeled with alkaline phosphatase (ALP). ALP activity is then measured with enzymatically amplified time-resolved fluorometry. The developed assay has many advantages over the radioactively labeled techniques previously used. In a preliminary clinical study involving 790 patient sera, we have identified 16 positive samples (2%). Highest titers were observed in a patient with melanoma and two breast cancer patients. Further studies are needed to improve the sensitivity of this test and to evaluate its possible use for cancer diagnosis, prognosis or monitoring of therapy.


An automated biotin-streptavidin procedure for progesterone evaluation. An automated biotin-streptavidin procedure for measuring progesterone in serum is described. The method is linear up to 98.3 nmol/L and the calibration curve is stable for at least 14 days. The lower limit of progesterone detection is 0.70 nmol/L. Although serum is the preferred specimen, progesterone levels can be measured in EDTA, heparin, or citrated plasma. There is no interference from samples with monoclonal proteins or from hemoglobin and bilirubin at concentrations of 9.62 g/L and 899 mumol/L, respectively. Lipemic samples will lower the progesterone levels. Total imprecision of the method in the range of 19-80.4 nmol/L gave CVs between 4.6 and 7.3%. Progesterone values obtained with this biotin-streptavidin procedure agreed with those obtained by the DPC RIA assay (r = 0.991). The biotin-streptavidin procedure can be used as an alternative to RIA for measurement of progesterone.


Congenital hypomagnesemia: alternatives to tissue biopsies for monitoring body magnesium status. We have been monitoring a 12-year-old boy (his present age) suffering from selective magnesium (Mg) malabsorption. After ascertaining his Mg status, we attempted to maintain Mg balance through the use of oral supplements. Plasma and erythrocyte Mg concentrations were monitored at bimonthly intervals. However these measurements did not accurately reflect Mg status and we subsequently measured Mg in the following tissues: lymphocyte, tooth, and hair. Levels of Mg in stable tissues such as temporary teeth and lymphocytes, unfailingly revealed a marked deficit in Mg that was only two-thirds of the normal levels found in the control group. The use of these readily-accessible stable tissues can thus obviate the need for muscle or bone biopsy. Unfortunately the level in hair is higher than in healthy subjects, and thus does not constitute an adequate measure of Mg status.


Apolipoprotein B gene polymorphism and plasma lipids and lipoproteins in a Canadian Caucasian population. We have investigated the frequency of Hind III DNA polymorphism of the human apolipoprotein B gene in a Canadian Caucasian population with coronary artery disease, as documented by angiography, and a healthy control population. Patients had significantly (p < 0.05) higher levels of cholesterol, triglycerides, LDL-cholesterol, apolipoprotein B, and lower level of apoAI compared to the controls. Restriction fragment-length polymorphism analysis detected nine hybridizable fragments denoted as H1 to H9. The H1, H2, H3, and H7 alleles were polymorphic. The [H4-H9] genotype seems to be the normal genotype within the population studied since it was detected in 69% of the control group. The [H1-H9] genotype was most frequently observed in the patients (frequency = 0.68). We were unable to strongly associate any of the alleles or genotypes detected with the changes in lipids. The additional alleles observed in the patient group may indicate possible mutations at the 3' end of the apolipoprotein B gene locus.