Alternative models for early onset of childhood leukaemia. This paper considers theoretical models for early-onset childhood leukaemia. The major focus of attention is the two-hit mutational model. A simple mathematical representation is used to explore mechanisms which might lead to onset of leukaemia at an unusually early age. Two such mechanisms are considered. The first of these, a germinal or very early embryonic first mutation is shown to imply that multiple independent leukaemic clones are likely to arise sequentially in very young patients. Clonal multiplicity could underlie the poor prognosis which has been associated with early onset childhood acute lymphoblastic leukaemia. It implies that curative therapy might require intensive treatment followed by bone marrow rescue to ensure eradication of all single-hit predisposed target cells. The prediction of multiple leukaemic clones might be tested in female patients by means of X-linked restriction fragment length polymorphisms and in patients with B-lineage neoplasms by determination of immunoglobin gene rearrangements. A second mechanism for early onset leukaemogenesis is the occurrence of a high cellular mutation rate in some patients. This is shown to result in leukaemia at significantly earlier age if the mutation rate is sufficiently high to influence target cell loss rate. This mechanism would enable more rapid clonal evolution of leukaemic cells and the early emergence of drug resistant variants. The prediction might be tested experimentally by sequential observation of genetic markers (e.g. Karyotypes, DNA fingerprint patterns) and the rate of emergence of drug resistant phenotypes. Other models, considered more briefly, include one-hit mutational 'dominants' in the developing embryo and faster growth kinetics in neoplasms of younger patients.(ABSTRACT TRUNCATED AT 250 WORDS)


The epidemiology of infant cancers.

The epidemiology of infant cancers. Cancers in infants represent less than 0.05% of all malignant neoplasms, but form a particularly interesting group for study. The ratio of solid tumours to leukaemias is 2:1 in children aged 1-14 but 5:1 in infants less than 1 year. The rate for neuroblastoma which is the most common malignancy in infants is four times higher in children aged under 1 year than in 1-14 year olds. Other embryonal tumours, e.g. Wilms', heptablastoma and retinoblastoma also show higher rates in infants. The ratios of incidence in males to females differed in a number of instances in the two age groups, e.g. in leukaemias and liver tumours the male to female ratio is greater than one in 1-14 year old children but less than one in infants. These observations suggest that many infant tumours may be aetiologically distinct. Their early onset and predominantly embryonal nature suggest a pre-natal origin and genetic factors may be important.


Cytogenetic findings in acute leukaemias of infants. Of 706 children, 528 with acute lymphoblastic leukaemia (ALL) and 178 with acute myelocytic leukaemia (AML), whose leukaemia karyotypes could be successfully analysed, 48 were infants less than 1 year of age, 28 with ALL (5% of ALL patients) and 20 with AML (11% of AML patients). In contrast to older children. ALL-leukaemocytogenetics in infants was characterised by lack of hyperdiploidy with over 50 chromosomes and higher incidence of pseudodiploidy. Thirteen (= 46%) infants had an 11q23 aberration, and 11 of them had t(4;11). In AML, nine (= 45%) infants also had an 11q23 abnormality, e.g. t(9;11). Thus, the 11q23 aberration was present in almost 50% of all leukaemia karyotypes of infants. In ALL of infants, the CALLA negative, pre-pre-B immunophenotype prevailed. In AML of infants, the monocytic subtype dominated. A biphenotypic morphology (lymphoid-monocytic) with the expression of lymphoid and myeloid antigens was seen in several ALL and AML cases. In conclusion, leukaemogenesis in infants is a rare event, arising in stem cells of very early hematopoietic differentiation (probably due to gene rearrangement errors, most frequently at FRA11B), and differs from leukaemogenesis in older age groups by unique clinical and cellular features.


Disposition of antineoplastic agents in the very young child. Maturation of physiologic process which govern the disposition of pharmacologic agents can yield significant changes in absorption, distribution, metabolism, and elimination of drugs in neonates, infants and children. However, there are very little data concerning the disposition of anticancer drugs in young children. Pharmacokinetic data for six anticancer agents were compared in infants less than 1 year of age and children greater than 1 year of age treated at St Jude Children's Research Hospital. No pharmacokinetic data were available for infants less than 2 months of age. Median methotrexate clearance tended to be lower in four infants (0.26-0.99 years) vs 108 children (1-19 years): 80 vs 103 ml min-1 m-2, respectively (P = 0.01). There was no difference in the median 42 h methotrexate concentration. Teniposide systemic clearance and terminal half-life and cytarabine systemic clearance were not different between the two groups. There was no significant difference in etoposide systemic clearance when normalised to body surface area (ml min-1 m-2), however a significantly lower systemic clearance relative to body weight (ml min-1 kg-1) was observed in two infants, 0.5 to 1 year of age, vs 23 children, 3-18 years of age. Doxorubicin systemic clearance was not significantly different between the two groups when systemic clearance was expressed in ml min-1 kg-1. However, there was a trend toward a lower rate of systemic clearance in ml min-1 m-2 in infants.(ABSTRACT TRUNCATED AT 250 WORDS)


The influence of age on nephrotoxicity following chemotherapy in children. Nephrotoxicity is an important adverse effect of chemotherapy in children. Renal function after treatment with either ifosfamide or cisplatinum in children aged 5 years or less ('younger children') was compared with that in those over 5 years ('older children'). Eighteen children (six younger, 12 older) given ifosfamide were studied after completion of chemotherapy, and 28 patients (16 younger, 12 older) were evaluated after cisplatinum. Glomerular filtration rate was measured from the plasma clearance of 51chromium-labelled edetic acid. Proximal tubular function was assessed by determination of plasma and urine calcium, phosphate, magnesium and glucose concentration; calculations of their fractional excretions, and of the renal threshold for phosphate; and measurement of urinary excretion of beta 2-microglobulin. Distal tubular function was evaluated by measurement of the early morning urine osmolality. Younger children had more severe proximal tubular toxicity than older children treated with ifosfamide, with significantly lower plasma phosphate concentrations and higher fractional excretions of glucose. However, there was no evidence of any such difference in glomerular or distal tubular damage after ifosfamide, and no difference in any aspect of renal function between younger and older children treated with cisplatinum. Increased proximal tubular toxicity after ifosfamide in younger children may have serious implications for future growth and development.


Brain and spinal tumours in children aged under two years: incidence and survival in Britain, 1971-85. There were 548 children aged under two with brain and spinal tumours diagnosed during 1971-85 in Great Britain and included in the population-based National Registry of Childhood Tumours. Children aged under two accounted for 12% of all children registered with tumours in these sites. The annual incidence was 25.0 per million. Ependymoma, astrocytoma and medulloblastoma (including primitive neuroectodermal tumour) each accounted for around a quarter of the total. Five-year survival rates were 20% for ependymoma, 43% for astrocytoma and 13% for medulloblastoma, each significantly lower than for children aged 2-14 in the corresponding diagnostic group. Mortality in the first year after diagnosis was very high but there were also substantial numbers of later deaths. There was no significant trend in survival rates during the period under review.


Leukaemia in the young child.

Leukaemia in the young child. Leukaemia is rare in infancy with an equal predominance of lymphoblastic and myeloblastic cases. Acute lymphoblastic leukaemia in infants under one year is characterised by a high leucocyte count, organomegaly, early B-cell phenotype, sometimes with evidence of monocytoid differentiation and cytogenetic abnormalities. This is reflected in its poor prognosis. The toddler (aged 1-2) tends to develop typical childhood ALL which is responsive to treatment, but remains vulnerable to late effects of therapy, particularly radiation. The distribution of subtypes of AML differs in the younger and older child and results of treatment have improved in all age groups. A uniform strategy appears desirable for all cases of childhood AML. It seems probable that different genetic and environmental factors may be involved in the genesis of infant ALL, childhood ALL and AML in children. The management of leukaemia in children under two poses a considerable challenge.


Children under two years treated according to the Medical Research Council UKALL VIII study and trial 1980-1984 (on behalf of the Medical Research Council Working Party on Leukaemia in Childhood). Ten per cent of children entered into the national leukaemia study UKALL VIII were under 2 years at diagnosis. The 6 year event-free survival of this cohort was 39%. Specific adverse features were age under 1 year, high initial white cell count and null cell ALL. Those with common ALL, WBC 10-50 x 10(9) 1-1 and especially those aged 18 months or older did not have an adverse prognosis compared with the whole trial entrants. Overall, however there was a doubling of CNS relapse rate and of both induction and remission deaths. Those with a WBC under 10 x 10(9) 1-1 had a high haematological relapse rate. The type of leukaemia and method of management rather than specifically the age appeared to be the predictor for poor outcome.


Acute myelogenous leukaemia in children under 2 years--experiences of the West German AML studies BFM-78, -83 and -87. AML-BFM Study Group. Clinical, morphological, immunological, cytogenetical and prognostic features of 84 children under 2 years of age with AML in studies AML-BFM-78, -83 and -87 were retrospectively analysed. There was a high incidence of acute monoblastic leukaemia (FAB M5) (41 patients--49%) and acute megakaryoblastic leukaemia (FAB M7) (study AML-BFM-87: five patients--13%) in this age group. Acute monoblastic leukaemia was associated with hepatosplenomegaly, extramedullary organ manifestations and chromosomal abnormalities involving 11q23. The probability of an 11-year event-free survival of all patients under 2 years of the three studies combined was 39% (SD 6%). While the event-free survival rates of patients aged 2 years and older could be improved in studies AML-BFM-83 and -87 compared with study AML-BFM-78, overall prognosis in children under 2 years in the three consecutive studies remained unchanged. The event-free survival rate of children with acute monoblastic leukaemia in both age groups was comparable (7 yr-EFS (AML-BFM-83 and -87): much less than 2 years--43% (SD 9%), =/much greater than 2 years--33% (SD 9%); P much greater than 0.5). This also applied to other risk groups. In conclusion, taking the high incidence of acute monoblastic and megakaryoblastic leukaemia in children under 2 years into account, no significant differences between children under 2 years or older children concerning response to therapy and overall prognosis could be evaluated.


Spontaneous regression of neonatal fibrosarcoma. We report a case of fibrosarcoma, which presented in a two week old boy. Excision was not performed because it would have required mutilating surgery. The tumour regressed and was impalpable by 7 months of age. The patient is tumour free at 4 years of age. This is the first reported case of spontaneous regression of a fibrosarcoma. The literature is reviewed. We conclude that the chances of metastasis are low, tumours are likely to respond to chemotherapy, and mutilating surgery is not appropriate initial treatment for infantile fibrosarcomas.


Allogeneic marrow transplantation in the treatment of infants with cancer. Marrow transplantation in infants with cancer present special challenges particularly because of the need to use conditioning regimens containing cytotoxic agents during a period of rapid somatic growth. An up-date of the largest study in children under 2 years of age treated by marrow transplantation for acute myeloblastic leukaemia, and a review of late effects experienced by older children, demonstrate the necessity to develop conditioning regimens which avoid total body radiation. Efforts to achieve this aim in the treatment of leukaemia are summarised.


Neonatal soft tissue tumours.

Neonatal soft tissue tumours. Thirty-five different soft tissue tumours occurring in the first month of life are described and classified into five Clinical Groups. A. Excellent prognosis with no treatment or simple surgical excision. B. Good prognosis. Treatment depends upon anatomical site. C. Good prognosis. Treatment usually surgical but chemotherapy may be indicated in certain situations. D. Intermediate prognosis. Treatment as for older child, usually surgery or chemotherapy. E. Poor prognosis. Treatment palliative or experimental. The relatively good prognosis of tumours in this age group and the importance of avoiding over-treatment are emphasised. Treatment guidelines are given and the value of obtaining tissue for biological studies to improve our understanding of these rare tumours is stressed.


The evaluation and management of pain in the infant and young child with cancer. In the last decade, there has been a developing awareness of pain in paediatric patients and especially in young patients who may be unable to express their pain. This paper addresses the issue of pain in the infant and young child with cancer. Current understanding of pain and its physiological consequences are explored. Evaluation of pain within a developmental context is outlined. Guidelines are presented for the behavioural and pharmacological management of pain with emphasis on appropriate therapy for the infant and young child.


Late effects of early childhood cancer therapy. The late effects of cancer treatment in children diagnosed early in life (under 2 years of age) may be compared to those in children who were over 2 years at the time of diagnosis. Such areas as growth and development (e.g., intellectual and sexual), vital organ function and risk for second cancer are of particular interest. This report reviews late occurring morbidity which was studied in approximately 400 survivors of childhood cancer, 93 of whom were under 2 years of age at diagnosis. The most commonly reported late effect was musculoskeletal in radiation treated patients. More severe cognitive deficits were seen among both age groups cranially irradiated for leukaemia prophylaxis with 24 Gy compared to 18 Gy. Second cancers developed equally between the two age groups. Predisposing factors and/or prior therapy may produce second cancers. There are inherent problems in assessing the outcomes of very young children treated for cancer. Improved survival has only been evident during the last 15 years, a period too short to appreciate many of the end points of interest in adult life. Such patients require a necessarily long follow-up period but this will prove informative in the future as more institutions initiate procedures for extended surveillance.


Poor prognosis neuroblastoma: is screening the answer? Neuroblastoma is one of the most common solid tumours of childhood and is unique amongst paediatric cancers in that it results in the urinary excretion of catecholamine metabolites which are easily measured in spot urine samples and so is a condition for which screening may be considered. The continuing poor prognosis associated with late stage disease has stimulated great interest in this proposition. The Japanese have been undertaking pioneering studies of such screening since 1974 and since 1985, all 6 months old babies have been offered screening. Preliminary data would appear to suggest that screening is effective in greatly improving the survival of children with neuroblastoma. However there are difficulties associated with the interpretation of survival data since screening undoubtedly results in the detection of cases which would otherwise have remained 'silent', and the well known problems of lead-time and length-time bias complicate matters still further. The time is not yet ready for universal implementation of screening and further investigation is required.


Using education to manage skill mix.

Using education to manage skill mix. The English National Board framework and higher national award for post-registration education has at its core the tripartite contract between practitioners, managers and educationalists. For managers the framework offers the potential to develop effective education and training strategies, particularly related to skill mix. By devising a skills matrix based on the ten characteristics of the framework, the author shows how managers can assess the skills required at unit, specialty and individual level.


Alzheimer's disease: does it exist?

Alzheimer's disease: does it exist? In posing the question that forms the title of this article, the author seeks not to provide a definitive answer, but to raise the issue in nursing literature. Through this, he suggests, nurses will become aware of the controversy that surrounds the diagnosis, or even existence, of Alzheimer's disease. His objective is to bring the nursing profession more fully into the arena where decisions are made about the future of research into the disease and appropriate strategies for the care of elderly people with dementia.


Strategic planning in patient education. Patient education is an important part of health promotion. It can be described as a planned, systematic process designed to influence the behaviour of those who come into contact with the health services. It produces changes in their knowledge, attitudes and the skills required to maintain and improve health. Patient education involves health care professionals in activities which help patients and their relatives learn and incorporate new behaviours into their everyday lives. The author describes a strategic planning tool aimed at achieving this.


Haemorrhagic brain injury: a care study. Mark was only 20 years old when an unfortunate sequence of events dramatically altered his life. In July 1989 he sustained two subarachnoid haemorrhages within a fortnight, first from a left anterior communicating artery aneurysm and then from a right middle cerebral artery aneurysm. Both aneurysm were successfully clipped but Mark remained hemiplegic with severe physical and behavioural problems, including incontinence, sexual disinhibition, aggression and uninhibited spitting. In November 1989, he was transferred to a neuro-rehabilitation unit and his management there will be described, showing how his complex problems were managed within enforced environmental limitations.


Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens. The relationships of 77 aerotolerant Arcobacter strains that were originally identified as Campylobacter cryaerophila (now Arcobacter cryaerophilus [P. Vandamme, E. Falsen, R. Rossau, B. Hoste, P. Segers, R. Tytgat, and J. De Ley, Int. J. Syst. Bacteriol. 41:88-103, 1991]) and 6 reference strains belonging to the taxa Arcobacter nitrofigilis, Arcobacter cryaerophilus, and "Campylobacter butzleri" were studied by using a polyphasic approach, in which we performed DNA-rRNA hybridizations, DNA-DNA hybridizations, a numerical analysis of whole-cell protein patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an analysis of cellular fatty acid compositions, and a phenotypic analysis and determined DNA base ratios. Our results indicate that "C. butzleri" should be transferred to the genus Arcobacter as Arcobacter butzleri comb. nov., as was suggested by Kiehlbauch and coworkers (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). A rapid screening of all strains in which we used the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique revealed five major groups, which were identified by using DNA-DNA hybridization data as A. cryaerophilus (two distinct electrophoretic subgroups), A. butzleri, A. nitrofigilis, and a new species, for which we propose the name Arcobacter skirrowii. The phylogenetic position within rRNA superfamily VI was established for each species. A. butzleri strains and strains belonging to one of the electrophoretic subgroups of A. cryaerophilus had similar fatty acid contents. An analysis of fatty acid compositions allowed clear-cut differentiation of all of the other groups. All of the species could be distinguished by using classical phenotypic tests, although erroneous identifications due to a shortage of clear-cut differentiating tests could occur.


Mycoplasma penetrans sp. nov., from the urogenital tract of patients with AIDS. An unusual mycoplasma, which was isolated from the urine of a human immunodeficiency virus-positive male homosexual patient, has an elongated flask shape and two unique sharply divided internal compartments. The tiplike compartment is densely packed with fine granules, and the body compartment is loosely filled with coarse granules consistent with ribosomal structures. The organism has properties of adherence, hemadsorption, and cytadsorption and invades many different types of mammalian cells. Adhesion and penetration apparently involve the terminally located tiplike structure. Cholesterol is required for growth, and the mycoplasma ferments glucose and hydrolyzes arginine, but does not hydrolyze urea. The results of DNA homology studies revealed that this organism is not genetically related to previously described mycoplasma species that have the same biochemical properties. The results of serologic studies demonstrated that this organism is antigenically distinct from all previously described mycoplasmas. We propose that this new mollicute species should be named Mycoplasma penetrans sp. nov. The type strain is strain GTU-54-6A1 (= ATCC 55252).


Enterococcus flavescens sp. nov., a new species of enterococci of clinical origin. Four yellow-pigmented group D enterococci of uncertain taxonomic position were isolated from several humans with severe infections. The results of DNA composition, DNA-DNA hybridization, fatty acid content, and biochemical property studies demonstrated that these organisms were slightly related to other previously described yellow-pigmented enterococcal species and constitute a new species, for which we propose the name Enterococcus flavescens. The type strain of E. flavescens is strain CCM 4239 [corrected].


Genomic fingerprinting by arbitrarily primed polymerase chain reaction resolves Borrelia burgdorferi into three distinct phyletic groups. The causative agent of Lyme disease, Borrelia burgdorferi, was first identified by Burgdorfer et al. in 1982 (W. Burgdorfer, A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davis, Science 216:1317-1319, 1982) and was isolated by Barbour et al. in 1983 (A. G. Barbour, W. Burgdorfer, S. E. Hayes, O. Peter, and A. Aeschlimann, Curr. Microbiol. 8:123-126, 1983). Since then, a large number of isolates have been collected, and there have been questions regarding the relationships among the various strains. Using genomic fingerprinting by an arbitrarily primed polymerase chain reaction, we resolved into three groups a collection of Eurasian and North American isolates of spirochetes that are generally categorized as B. burgdorferi. Group I strains have been identified in both North America and Eurasia, while strains belonging to Borrelia groups II and III have been found only in Eurasia. These same three groups have also been delineated by Baranton et al. (G. Baranton, D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont, Int. J. Syst. Bacteriol. 42:370-375, 1992) by independent methods. Two isolates are distinct from all of the other strains in our collection but are clearly members of the genus Borrelia.


Legionella shakespearei sp. nov., isolated from cooling tower water. A Legionella-like organism (strain 214T [T = type strain]) was isolated from a cooling tower in Stratford-upon-Avon, England. This strain required L-cysteine and contained cellular branched-chain fatty acids that are typical of the genus Legionella. Strain 214T produced pink colonies on buffered charcoal-yeast extract agar. Ubiquinone Q-12 was the major quinone. Strain 214T was serologically distinct from other legionellae as determined by a slide agglutination test. The results of DNA hybridization studies showed that strain 214T (= ATCC 49655T) is a member of a new Legionella species, Legionella shakespearei.


Description of Porphyromonas circumdentaria sp. nov. and reassignment of Bacteroides salivosus (Love, Johnson, Jones, and Calverley 1987) as Porphyromonas (Shah and Collins 1988) salivosa comb. nov. A new species, Porphyromonas circumdentaria, is proposed for pigmented, asaccharolytic strains that were isolated from the gingival margins or mouth-associated diseases of cats. This bacterium is an obligately anaerobic, gram-negative, brown- or black-pigmented, asaccharolytic, nonmotile, nonsporing, rod-shaped organism which does not grow in bile and has a guanine-plus-cytosine content of 40 to 42 mol%. It produces major amounts of acetic, butyric, and isovaleric acids and minor amounts of propionic, isobutyric, and phenylacetic acids as end products of metabolism in cooked meat medium. Glutamate and malate dehydrogenases are present, while 6-phosphogluconate and glucose-6-phosphate dehydrogenases are absent. The major cellular fatty acid is 13-methyltetradecanoic acid (iso-C15:0 acid). P. circumdentaria strains are catalase positive and produce ammonia, and colonies fluoresce under short-wavelength UV light. These strains do not hemagglutinate erythrocytes, exhibit trypsinlike activity, or produce chymotrypsin or alpha-fucosidase. They are heavily piliated and produce a capsule. The type strain is strain VPB 3329 (= NCTC 12469). Bacteroides salivosus (D. N. Love, J. L. Johnson, R. F. Jones, and A. Calverley, Int. J. Syst. Bacteriol. 37:307-309, 1987) is an obligately anaerobic, gram-negative, pigmented, asaccharolytic, nonmotile, rod-shaped organism which does not grow in bile and has a guanine-plus-cytosine content of 42 to 44 mol%. This organism produces major amounts of acetic, butyric, and phenylacetic acids and minor amounts of isobutyric and isovaleric acids as end products of metabolism in cooked meat medium.(ABSTRACT TRUNCATED AT 250 WORDS)


Flexibacter ovolyticus sp. nov., a pathogen of eggs and larvae of Atlantic halibut, Hippoglossus hippoglossus L. A psychrotrophic Flexibacter sp., Flexibacter ovolyticus sp. nov., was isolated from the adherent bacterial epiflora of Atlantic halibut (Hippoglossus hippoglossus L.) eggs and was shown to be an opportunistic pathogen for halibut eggs and larvae. The strains which we isolated had the enzymatic capacity to dissolve both the chorion and the zona radiata of the egg shells. A total of 35 isolates were characterized by using morphological and biochemical tests. These strains were rod shaped, gram negative, Kovacs oxidase positive, and pale yellow and exhibited gliding motility. They did not produce acid from any of the wide range of carbohydrates tested. Our isolates had the ability to degrade gelatin, tyrosine, DNA, and Tween 80. Starch, cellulose, and chitin were not degraded. The strains were catalase and nitrate reductase positive, did not produce H2S, and did not grow under anaerobic conditions. F. ovolyticus resembles Flexibacter maritimus, but differs from the latter species in several biochemical and physiological characteristics. DNAs from F. ovolyticus strains had guanine-plus-cytosine contents which ranged from 30.3 to 32.0 mol% (strains EKC001, EKD002T [T = type strain], and VKB004), and DNA-DNA hybridization studies revealed levels of relatedness between F. ovolyticus EKD002T and F. maritimus NCMB 2154T and NCMB 2153 of 42.7 and 30.0%, respectively. Compared with previously described Cytophaga and Flexibacter spp. with low guanine-plus-cytosine contents, F. ovolyticus constitutes a new species. Strain EKD002 (= NCIMB 13127) is the type strain of the new species.


DNA relatedness among Erysipelothrix rhusiopathiae strains representing all twenty-three serovars and Erysipelothrix tonsillarum. The levels of relatedness among strains of Erysipelothrix rhusiopathiae (serovars 1 through 23 and type N) were estimated by performing DNA-DNA hybridization experiments with the type strains of E. rhusiopathiae and Erysipelothrix tonsillarum, which are the two Erysipelothrix species that have been described. Two distinct DNA relatedness groups were identified. The group 1 strains, representing serovars 1, 2, 4 through 6, 8, 9, 11, 12, 15 through 17, 19, and 21 and type N, exhibited more than 73% hybridization with the type strain of E. rhusiopathiae but less than 24% hybridization with the type strain of E. tonsillarum. Group 2 included serovar 3, 7, 10, 14, 20, 22, and 23 strains, and these strains exhibited more than 66% hybridization with the type strain of E. tonsillarum but less than 27% hybridization with the type strain of E. rhusiopathiae. Strains representing serovars 13 and 18 exhibited low levels of hybridization (16 to 47%) with both of the type strains, indicating that these serovars may be members of a new genomic species. The members of the E. rhusiopathiae and E. tonsillarum groups resembled each other in many phenotypic characteristics, but differed in their ability to produce acid from saccharose and in their pathogenicity for swine. Our results confirm that the genus Erysipelothrix contains two main genomic species, E. rhusiopathiae and E. tonsillarum, which can be differentiated into serovars.


Chemotaxonomic differentiation of coryneform bacteria isolated from biofilters. Coryneform bacteria that were isolated from biofilters which are used for waste gas treatment of animal-rendering plant emissions were differentiated and partially identified by using chemotaxonomic methods. On the basis of the results of a numerical analysis of whole-cell fatty acid profiles, 79 isolates were divided into two major groups; the members of the first group contained saturated and monounsaturated fatty acids, whereas the members of the second group were characterized by iso- and anteiso-branched fatty acids. Division into subclusters was based mainly on quantitative differences in fatty acid composition and was confirmed by the results obtained for additional chemical markers (e.g., respiratory quinones, mycolic acids, polar lipids, cell wall amino acids, and whole-cell sugar patterns). By combining the results obtained for chemotaxonomic analyses that were performed for strains containing saturated and monounsaturated fatty acids, we were able to identify the genus Corynebacterium (two Corynebacterium species were differentiated on the basis of the occurrence of tuberculostearic acid), the genus Gordona, and the genus Mycobacterium. Among the strains that produced iso-anteiso fatty acid patterns, one subgroup was affiliated with the "nicotianae" group of the genus Arthrobacter; however, some strains contained a new combination of chemical markers. Peptidoglycan type A4 alpha, L-Lys-Gly-L-Glu was combined with menaquinones MK-7 and MK-8, whereas peptidoglycan type A4 alpha, L-Lys-L-Glu occurred together with MK-8 and MK-9. The second subgroup was characterized by a new type B peptidoglycan and MK-11, as well as small amounts of MK-12. Differentiation that was based first on chemotaxonomy and second on physiology gave reliable results. Thus, coryneform strains with new characteristics were isolated from biofilters.


Taxonomic study of the Lactobacillus acidophilus group, with recognition of Lactobacillus gallinarum sp. nov. and Lactobacillus johnsonii sp. nov. and synonymy of Lactobacillus acidophilus group A3 (Johnson et al. 1980) with the type strain of Lactobacillus amylovorus (Nakamura 1981). Biochemical properties and DNA-DNA reassociation studies of Lactobacillus acidophilus strains isolated from humans and animals indicate that these include six genomospecies. Two new species can be differentiated from the established species of the genus Lactobacillus: L. gallinarum sp. nov. (type strain, ATCC 33199) and L. johnsonii sp. nov. (type strain, ATCC 33200). Furthermore, it was clarified that L. acidophilus group A3 (Johnson et al. 1980) is synonymous with L. amylovorus.


Tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction by mycoplasmas. We investigated 22 mycoplasma and acholeplasma species for their ability to reduce tetrazolium salts by using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The test results were evaluated visually, as well as spectrophotometrically, by using an enzyme-linked immunosorbent assay reader. Our results were very similar to the results obtained when the tetrazolium salt reduction assay described by Aluotto et al. was used. However, the MTT reduction assay appeared to be better because it is faster, more objective and sensitive, easier to evaluate, and less expensive; in addition, it allows quantitative determinations. By using regression analysis a linear correlation between formazan production and the number of colony-forming units was demonstrated for all of the species investigated, indicating that the MTT assay can also be used for growth, toxicity, or chemosensitivity tests for the mycoplasma species that are capable of reducing tetrazolium salts.


Detection of drugs in human hair for clinical and forensic applications. While hair has for sometime been analyzed for assessment of trace elements, it is only in recent years that attention has been focused on this matrix as a possible means of evaluating drug impregnation. This technique was applied to treated subjects and drug abusers for determination of drug consumation. The method involved decontamination in ethanol, solubilization in sodium hydroxide at 100 degrees C for 10 min, extraction in chloroform/isopropanol/n-heptane (50:17:33, v/v), separation on a BP-5 capillary column (GC) and detection by mass spectrometry. Hair samples were analysed for barbiturates, antidepressants, benzodiazepines, beta-blockers, nicotine, opiates, benzoylecgonine, cannabis and amphetamines.


Technical parameters influencing the severity of injury of front-seat, belt-protected car passengers on the impact side in car-to-car side collisions with the main impact between the front and rear seats (B-pillars). Authentic car-to-car side collisions (n = 30) with the main impact area at the B-pillar were analyzed to find technical parameters corresponding with the injury severities of the front seat, belt-protected car passengers on the impact side. EES (Energy Equivalent Speed) and delta v (delta v, change in velocity) were highly significant predictors of the severity of thoracic and abdominal injuries and total injury severity coded according to the Abbreviated Injury Scale (AIS). At an EES or delta v greater than or equal to 40 km/h all front-seat car passengers on the impact side sustained a total injury severity of Maximum AIS (MAIS) greater than or equal to 4 and died. Although a passenger could survive the crash without injury to one or more body regions up to the highest EES- and delta v-values, at EES or delta v greater than or equal to 40 km/h fatal injuries were sustained in at least one body region. At an EES greater than or equal to 35 km/h or a delta v greater than or equal to 15 km/h no front-seat car passenger on the impact side remained uninjured.


Characterisation of HLA DQ alpha for forensic purposes. Allele and genotype frequencies in British Caucasian, Afro-Caribbean and Asian populations. Allele and genotype frequencies for British Caucasian, Afro-Caribbean and Asian populations were determined for a total of over 600 unrelated individuals at the HLA-DQ alpha locus. These were analysed by polymerase chain reaction amplification of the DNA followed by hybridisation to allele specific oligonucleotide probes in a reversed dot-blot test. Six different alleles were detected and the allele distributions for the 3 populations analysed displayed significant differences. However, the British Caucasian genotypes were statistically very similar to previously published data from US Caucasians as were British Afro-Caribbean genotype frequencies with US Black data. In Caucasians the allele frequencies ranged from 5.2% to 26.9% with a power of discrimination of 0.93. DQ alpha genotype frequencies of Caucasian and Afro-Caribbean populations do not deviate from Hardy-Weinberg equilibrium. However, the Asian data displayed significant deviation due to excess homozygotes.


Immunohistochemical localization of fibronectin as a tool for the age determination of human skin wounds. We analyzed the distribution of fibronectin in routinely embedded tissue specimens from 53 skin wounds and 6 postmortem wounds. In postmortem wounds a faint but focal positive staining was exclusively found at the margin of the specimens which did not extend into the adjacent stroma. Vital wounds were classified into 3 groups. The first comprising lesions with wound ages ranging from a few seconds to 30 min, the second comprising those with wound ages up to 3 weeks, and the third group with lesions more than 3 weeks old. Ten out of 17 lesions with a wound age up to 30 min showed a clear positive reaction within the wound area. Three specimens in this group were completely negative, while in 4 additional cases the result was not significantly different from postmortem lesions. These 7 cases were characterized by acute death with extremely short survival times (only seconds). In wounds up to 3 weeks old fibronectin formed a distinct network containing an increasing number of inflammatory cells corresponding to the wound age. In 2 cases with a survival time of 17 days and in all wounds older than 3 weeks fibronectin was restricted to the surface of fibroblasts and to parallel arranged fibers in the granulation tissue without any network structures. We present evidence that fibronectin is a useful marker for vital wounds with a survival time of more than a few minutes. Fibronectin appears before neutrophilic granulocytes migrate into the wound area.(ABSTRACT TRUNCATED AT 250 WORDS)


A suicidal death by explosives.

A suicidal death by explosives. A 23-year-old man committed suicide by detonating an explosive substance clutched to his abdomen. The face was singed, the hands were lacerated and the chest and the upper part of abdomen were badly mutilated. The death was due to the destruction of several vital organs. The injuries had a directional nature. Only the consideration of all the facts of the case in relation to the situation, nature, distribution and extent of wounds and the preceding medical and social history allowed a reconstruction of the circumstances.


DNA fingerprinting of freeze-dried tissues. DNA profiling is usually unproblematic when carried out on material sampled shortly after death. Prolonged postmortem intervals and improper storage cause significant DNA degradation, however there are instances where DNA analysis becomes necessary months after the autopsy. In such cases the investigator may be able to revert to material initially stored for toxicological purposes. In some cases such specimens undergo freeze-drying before storage. We have therefore tested DNA fingerprinting of freeze-dried postmortem material. It was found that freeze-drying is a suitable method for preserving tissue samples for DNA profiling.


Sudden death due to disseminated porocephalosis--a case history. An 18-year-old Nigerian girl died suddenly and unexpectedly from disseminated porocephalosis. The patient was hospitalised complaining of fever, dizziness, weakness and jaundice. Clinical examination revealed a restless, confused and hypotensive patient. She died within two hours of admission. Autopsy revealed disseminated Porocephalus armilatus infestation involving the thoracic and abdominal linings and their internal organs. The relationship between the disseminated parasitic involvement and the death of this girl is discussed.


Biostatistical evaluation of blood group, HLA and DNA findings in Jeffreys' immigrant test-case using special kinship and DNA algorithms. In the immigration case cited by Jeffreys et al. (1985), the biostatistical evaluation of blood group findings in 16 systems and of HLA-A,B findings for the mother and child, using a special kinship algorithm developed by Ihm and Hummel (1975), produced a probability of maternity of the Ghanaian-born putative mother of W = 6%; the probability of maternity for her sister was W = 94%. Using the DNA multilocus probes 33.15 and 33.6 and the bandsharing technique, the authors analysed band patterns from the putative mother and child as well as another 3 children of the same woman. It was concluded that the putative mother was the mother of all 4 children. An evaluation of the band patterns using the multi-di-allelism model and the kinship algorithm in accordance with the Essen-Möller principle produced: W = 99.99999999999999991%, or, if the "-----" constellations were not considered, W = 99.99998%.


Suppression of ischemia-reperfusion injury by liposomal superoxide dismutase in rats subjected to tourniquet shock. To investigate the role of oxygen-derived free radicals in the pathogenesis of tourniquet shock, the authors present an experimental animal model. Two groups of rats were fastened with rubber tubes on both thighs (1.5 kg/cm2) for 6 h under pentobarbital anaesthesia. One group was administered liposomal superoxide dismutase (L-SOD 30,000 U/kg body weight), and the other liposome as a control 3 h prior to tourniquet removal. No rats in the control group (n = 20) survived more than 24 h after reperfusion, whereas 55% of animals treated with L-SOD (n = 20) survived for 24 h or more, and two recovered completely (P less than 0.005). Blood samples were obtained from the abdominal aorta after laparotomy of anaesthetized rats of both groups at different time intervals. Changes in the hematocrit value and blood urea nitrogen during the early periods after reperfusion were attenuated by prior administration of L-SOD, and the total plasma SOD activity of the control animals decreased promptly and continuously throughout the experimental period. This experimental model was very useful to study the pathogenesis of tourniquet shock with respect to reproducibility, induction of the shock stages and mortality. It is thought that oxygen-free radicals are involved in the induction of tourniquet shock, and L-SOD was, to a certain extent, effective against reperfusion injury in the early stages of shock.


Formal and population genetic studies of AHSG: further data from Galicia. A sensitive immunodetection method for Alpha-2-HS glycoprotein (AHSG) after ultrathin layer polyacrylamide gel isoelectric focusing has been applied to a family study of 126 matings including 292 offspring. Formal genetic studies are in agreement with an autosomal mode of inheritance for this system. A population study of 506 unrelated individuals from Galicia (NW Spain) gave the following frequencies: AHSG*1 = 0.7559 and AHSG*2 = 0.2441, which correspond to a exclusion chance for non-fathers of 0.1505.


Bite injuries upon a newborn.

Bite injuries upon a newborn. A 2 1/2-year-old boy inflicted on his 7-day-old brother suction and bite injuries of the head with loss of tissue of nose and lips. Place of the incident was the parents' bed. The event motivated us to report on the assignment of such injuries and the reasons for sibling violence.


Differential regulation and polyadenylation of transferrin mRNA in Xenopus liver and oviduct. Estrogen destabilizes transferrin mRNA in male Xenopus liver in the same manner as observed for albumin and gamma-fibrinogen. The present study examined estrogen regulation of transferrin gene expression in female Xenopus liver and oviduct. In female Xenopus liver estrogen causes the same enhanced degradation of transferrin mRNA from the cytoplasm as seen in males. In contrast, transferrin is induced 3- to 4-fold in both oviduct nuclear and cytoplasmic RNA. The similar increase in transferrin RNA in both preparations suggests a transcriptional mechanism is responsible for this stimulation. Therefore, transferrin expression is differentially regulated in these tissues by the same hormone. Previous experiments showed that Xenopus serum albumin mRNA has a very short (17 residue) poly(A) tail that may play a role in its hormone-regulated instability. Transferrin mRNA has a similarly short poly(A) tail in liver of both male and female Xenopus. Estrogen has no effect on transferrin polyadenylation in liver. Similarly short poly(A) is found on transferrin mRNA from estrogen-deprived oviducts in explant culture. However, addition of estradiol to the medium results in the appearance of a 50-200 nucleotide poly(A) concurrent with induction. Therefore, transferrin mRNA is differentially polyadenylated in Xenopus liver and oviduct. In the latter tissue polyadenylation is under hormonal control.


The metabolic pathways for hormonal steroids appear to be reflected in the stereochemistry of DNA. Computer graphics and energy calculations were employed to examine the relative fit of progesterone and its major biosynthetic precursors and inactive metabolites into partially unwound double stranded DNA. Progesterone was found to be the best fitting molecule; moreover, it was the only compound which exhibited full stereochemical complementarity by inserting completely between base pairs and forming optimal hydrogen bonds with both deoxyribose-phosphate backbones. Intermediates in each step of the biosynthetic and degradation pathways were progressively increasing and decreasing fits into DNA, respectively. When the fits of various possible stereoisomers were examined, the positions of functional groups manifest in the known biosynthetic precursors were found to provide the best possible fitting structures. Conversely, the positions of functional groups of known inactive metabolites provided the worst possible fitting structures. These findings coupled with previous reports showing that the specific biological function assigned to each class of steroid hormone correlates with the formation of a unique pattern of donor/acceptor linkages confirms that hormonal structures are indeed rare in their capacity to form "lock and key" complexes with DNA. Given that all possible linkages to DNA are not yet accounted for, the existence of other naturally occurring compounds with salient biological function is predicted.


A Leu----His substitution at residue 93 in human corticosteroid binding globulin results in reduced affinity for cortisol. A steroid binding capacity assay and a radioimmunoassay were both used to measure corticosteroid binding globulin (CBG) in serum samples from 22 patients with sepsis. An approximately 50% discordancy between the two values in one patient suggested the presence of a CBG variant with reduced affinity for cortisol, and this was confirmed by Scatchard analysis. We therefore used the polymerase chain reaction to amplify exons that encode for human CBG from the genomic DNA of this patient. This revealed two mutations within the coding sequences: one of which results in a Leu----His substitution at residue 93 and another which encodes a Ser----Ala substitution at residue 224 of the human CBG polypeptide. To assess the impact of each substitution on the steroid binding affinity of CBG, each mutation was introduced separately into a normal human CBG cDNA, and the normal and mutated cDNAs were expressed in Chinese hamster ovary cells. Scatchard analysis of the CBG produced in culture indicated that the His93 mutation (Kd = 2.24 +/- 1.75 nM) reduced the cortisol binding affinity of CBG (mean +/- SD) significantly (P less than 0.024) when compared to normal CBG (Kd = 0.64 +/- 0.31 nM), while the Ala224 mutation (Kd = 0.63 +/- 0.33 nM) did not influence cortisol binding affinity. We therefore conclude that residue 93 may play an important role in determining the structure of the CBG steroid binding site.


Novel antiprogestins Org 31806 and 31710: interaction with mammalian progesterone receptor and DNA binding of antisteroid receptor complexes. We have examined steroid binding parameters and transformation of calf uterine progesterone receptor (PR) liganded with progestins (progesterone and R5020) and the newly synthesized antiprogestins (Org 31806 and 31710). Species specificity analysis indicated that [3H]R5020 binding in the chicken oviduct cytosol could be eliminated in the presence of 100-fold excess radioinert progesterone and R5020 but not Org 31806 and 31710. In the calf uterine cytosol, the progestins and the antiprogestins appeared to interact with the same PR as revealed by the displacement of [3H]R5020 by all of the above steroids. When the extent of [3H]R5020 binding was examined in the presence of different concentrations of radioinert steroids, the relative affinity with which these compounds interacted with the uterine PR was found to be comparable. A 23 degrees C incubation of cytosol transformed the progestin-bound PR complexes increasing their binding to DNA-cellulose from 5 (0 degrees C, nontransformed) to 35%. Under these conditions, 20% Org 31710- and RU486-occupied PR complexes bound to DNA-cellulose whereas only 10% Org 31806-receptor complexes were retained by the resin. Transformation (23 degrees C) of cytosol receptor caused a loss of the larger 8 S form and an increase in the smaller 4 S form. In its unliganded state or when it was complexed with R5020 or the antiprogestins, incubation of PR at 23 degrees C led to dissociation of the receptor-associated 90 kDa heat-shock protein (hsp90). The PR-hsp90 association was stabilized in the presence of 10 mM iodoacetamide when the ligand binding site was occupied by Org 31806 and 31710. The R5020-receptor complexes, however, allowed release of hsp90 under the above transforming conditions. Our results indicate that although Org 31806 and 31710 show no affinity for the avian PR, these steroids interact with the mammalian PR. We propose that the reported antiprogestational effects of Org 31806 and 31710 are mediated via their interaction with PR which appears similar to one that exists between PR and RU486.


Properties of a potent LHRH antagonist (Org 30850) in female and male rats. Org 30850 (Ac-D-pClPhe1,2,D-Bal3,D-Lys6,D-Ala10-LHRH) is a novel LHRH antagonist, which is being developed for the treatment of hormone-dependent disorders. The activities of this compound with respect to its endocrinological properties and side-effects were tested in rats and the results were compared with one of the first LHRH antagonists: Ac-D-pClPhe1,2,D-Trp3,D-Arg6,D-Ala10-LHRH (Org 30276). A single subcutaneous (s.c.) dose of 0.3 micrograms/kg Org 30850 administered to rats in pro-estrus gave inhibition of ovulation in approx. 50% of the rats, whereas Org 30276 was approx. 4 times less potent. The effect of a single s.c. injection of Org 30850 on testosterone levels in young adult male rats was also studied. The administration of 250 micrograms/kg or higher of Org 30850 induced a significant decrease in testosterone levels after 3 h, this effect lasted for at least 48 h. Treatment of female rats for 14 days with a daily dose of 12 micrograms/kg Org 30850 decreased statistically significantly uterine and ovarian weights. At a daily dose of 50 micrograms/kg Org 30850 completely suppressed estrous cycles and significantly decreased estradiol and FSH serum levels. The LH levels were below the detection level in both control and treated animals on the (expected) second day of di-estrus. Treatment of male rats for 14 days (25-200 micrograms/kg) resulted in a dose-dependent reduction of the gonads, accessory sex organs, testosterone levels and gonadotrophins. The decrease in gonadal function in both sexes was reversible since the females proved to be as fertile as the controls 6 weeks after the last treatment and an almost complete recovery of the weight of testes, seminal vesicles and ventral prostate was observed in the males 4 weeks after cessation of treatment. In contrast to Org 30276, Org 30850 exerted very slight irritation at the site of injection and no edematous reactions in the extremities at a daily dose of up to 8 mg/kg in male rats. It is concluded that Org 30850 is a very potent LHRH antagonist without edematous reactions and with a more favourable therapeutic index than Org 30276.


Identification of an estrogen-binding protein in Pseudomonas aeruginosa. A constitutive estrogen-binding protein (EBP) has been identified in the cytosol of Pseudomonas aeruginosa, a Gram-negative bacterium. All 14 strains tested contained the EBP. Estradiol binding was rapid and maximal binding occurred by 90 min at 0 degrees C. Dissociation of estradiol from the binding protein occurred at a rate of 4.6 fmol/min with a t1/2 of 42 min. EBP binding was destroyed by protease treatment and at high temperature. Sodium molybdate had no effect on binding. The Kd determined by Scatchard analysis was 3.9 nM and the Bmax was 323 fmol/mg protein. The EBP sedimented at 8.9 S on sucrose density gradients. The presence of 0.4 M KCl increased estradiol binding 6-fold but did not cause a shift in the sedimentation value. Gel filtration of the native protein gave an estimated molecular weight of 215,000 and a Stokes radius of 50.2 A. Steroid binding specificity, in order of decreasing affinity, was estradiol, estrone, dihydrotestosterone, estriol, testosterone, progesterone and promegestone. Other steroid hormones tested did not compete for estradiol binding. Identification of an EBP in a bacterium allows a comparative analysis of other steroid-binding proteins in unicellular microorganisms.


The influence of NADPH-dependent lipid peroxidation on the progesterone biosynthesis in human placental mitochondria. In an in vitro system consisting of human term placental mitochondria and an NADPH-generating system plus Fe2+, significant lipid peroxidation was observed along with a concomitant inhibition of progesterone biosynthesis. This inhibition could be markedly blocked by Mn2+, superoxide dismutase and dimethylfuran, inhibitors of NADPH-dependent lipid peroxidation. In addition, it has been found that malondialdehyde formation is accompanied by a corresponding decrease in placental mitochondrial cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in cell-free systems. These measurements provide the first evidence that the inhibition of progesterone biosynthesis by a NADPH-dependent lipid peroxidation in placental mitochondria is a consequence of cytochrome P-450 degradation due to lipid peroxidation.


Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles--I. Binding and transport. To gain insight into the mechanisms governing cellular uptake of glucocorticoids, we studied the binding and membrane transport of corticosterone (B) on a highly purified plasma membrane fraction from rat liver that was homogenized using a gentle, isotonic procedure. The fraction was mostly in the form of right-side out and osmotically active vesicles that were free of intracellular glucocorticoid receptors (GCR), transcortin (CBG) and ATP. Our uptake and binding studies carried out at 22 degrees C with [3H]B in physiological concentrations resulted in the following findings: (1) unlabeled B competed with [3H]B for uptake by the membrane vesicles; half-maximal competition of specific uptake was achieved with a 10- to 11-fold molar excess of unlabeled B. (2) [3H]B uptake was a saturable process of unusual kinetics (multiple sigmoidity); modified Scatchard plots revealed three significantly different apparent Kd-values of 1.3, 4.7 and 17.3 nM, corresponding to free B in the blood of non-stressed rats (4-16 nM). (3) Osmotic shrinkage of the vesicles led to a linear decrease in specific uptake, while non-specific uptake was independent of vesicle volume. Passive diffusion of [3H]B took place in leaky, but not in intact, vesicles. Reversible binding to, and mediated transport through, the membrane were interdependent parts of a strongly linked process. B was accumulated inside the vesicle up a concentration gradient by an active transport that followed first-order kinetics (Kt:3.9 nM); for its statistically reliable mathematical formulation and kinetic analysis, a replot was developed that revealed that relative accumulation increased with decreasing external hormone concentration. (4) Comparative binding studies disclosed that the apparent Kd-values (86.5 +/- 7.3 and 77.0 +/- 14.3 nM, respectively) of the [3H]B interactions with CBG and GCR did not differ (P greater than 0.3). These findings permit the conclusion that a plasma membrane-inserted carrier for B, effectively operating at physiological concentrations in the blood, is involved in a functional and regulatory manner in the biological action of glucocorticoids.


Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles--II. Comparative influx and efflux. To elucidate the initial step in the interaction between glucocorticoids (GC) and the hepatocyte, we examined at 22 degrees C further kinetic properties of active corticosterone (B) transport mediated by a putative, plasma membrane-inserted carrier for GC (GCC) as previously reported [Alléra and Wildt, J. Steroid Biochem. Molec. Biol. 42 (1992) 737-756]. We used a purified, well-characterized, osmotically active vesicle fraction of plasma membrane (PM), free of ATP, isolated from rat liver and a method developed by us to describe transport processes mathematically: (1) uptake (U) of 7 nM B into the vesicles (influx, I) occurred very rapidly whereby T1/2 = 8.3 s, the time (S) required for half maximum transport; the influx velocity (dU/dS = V) decreased degressively with time following second-order kinetics characterized by an initial transport V (VT0) of 177.7 fmol/mg membrane protein/s. (2) VToI of B-influx rose with temperature biphasically (P less than 0.025): activation energy above and below 15 degrees C (at PM phase transition) amounted to 9.5 and 26.5 kJ/mol. Neither at 45 nor at 60 degrees C did transport take place, revealing the high thermolability of GCC. (3) Efflux (E) of 6.5 nM B, i.e. transport out of the vesicles preincubated with the steroid, showed that influx had resulted in a 19.6-fold intravesicular hormone accumulation, indicating active ("uphill") transport. (4) The efflux velocity (dE/dS = V) exhibited almost the same kinetic quality as that of influx: it decreased following mainly second-order kinetics whereby T1/2 = 8.0 s. However, its whole time-course was much slower and the VT0 of efflux (VToE) was 6.3 lower than VToI. (5) Using physics and thermodynamics, we deduced that the affinity (AF) between B and GCC is proportional to the square of VT0. (6) Thus, because AF approximately (1/6.3)2, AF of the B-GCC interaction after completion of influx was calculated to be 40 times lower (Kd = 708 nM; delta G degrees = -34.9 kJ/mol) than at outset of influx, whereby delta G degrees = -44.0 kJ/mol. Concluding from these and previous findings, we present a new hypothesis on B transport into the hepatocyte: There is no difference (P greater than 0.3) in free enthalpy between transcortin (CBG) and the intracellular GC receptor interacting with B (delta G degrees = -40.1 and -40.4 kJ/mol). The GCC, however, is characterized by its ability to switch from a high- to lower-affinity when interacting with B (and vice versa due to metabolic energy input).(ABSTRACT TRUNCATED AT 400 WORDS)


Changes in plasma testosterone levels during the peri-hatching period in the chicken. Blood was obtained by heart puncture from 19-day-old Black Sex link chicken embryos and from Black Sex link chickens at 1.5, 6, or 24 h post-hatching. Plasma testosterone was determined by gas chromatography-mass spectrometry associated with stable isotope dilution. At 19 days the plasma of male and female chick embryos contains measurable amounts of testosterone and levels do not differ between sexes. After hatching plasma testosterone gradually declines from pre-hatch concentrations in males and females, but in all the post-hatch ages studied, plasma testosterone was significantly higher in male than in female chicks. These results indicate that in male chickens, contrary to mammals at birth, there is no surge in plasma testosterone at hatching.


Transforming growth factor beta modulates phosphorylation of the epidermal growth factor receptor and proliferation of A431 cells. Transforming growth factor beta (TGF-beta) increased the phosphorylation of the epidermal growth factor (EGF) receptor and inhibited the growth of A431 cells. Incubation with TGF-beta induced maximal EGF receptor phosphorylation to levels 1.5-fold higher than controls. Phosphorylation increased more prominently (4-5-fold) on tyrosine residues as determined by phosphoamino acid analysis and antiphosphotyrosine antibody immunoblotting. The kinase activity of EGF receptor was also elevated 2.5-fold when cells were cultured in the presence of TGF-beta. The antiproliferative effect of TGF-beta on A431 cells was accompanied by prolongation of G0-G1 phase and by morphological changes. TGF-beta augmented the growth inhibition of A431 cells which could be induced by EGF. In parallel, the specific EGF-induced increase in total phosphorylation of the EGF receptor was also augmented in the presence of TGF-beta. In cells cultured with TGF-beta, the phosphorylation of EGF receptor tyrosines induced by 20-min exposure to EGF was further increased 2-3-fold, suggesting additive effects upon receptor phosphorylation. EGF receptor activation by TGF-beta is characterized by kinetics quite distinct from that induced by EGF and therefore appears to take place through an independent mechanism. The TGF-beta-induced elevation in the phosphorylation of the EGF receptor may have a role in the augmented growth inhibition of A431 cells observed in the presence of EGF and TGF-beta.


Stability of retinoblastoma gene expression determines the tumorigenicity of reconstituted retinoblastoma cells. Mutational inactivation of the retinoblastoma gene (RB) is an invariant feature of the childhood eye cancer retinoblastoma and of tumor cells derived therefrom. In a previous study, retrovirus-mediated transfer of wild-type RB into cultured retinoblastoma cells resulted in a marked enlargement and reduced growth rate of these cells, as well as loss of their tumorigenic properties in nude mice. It was therefore difficult to separate the proposed growth-suppressing and tumor-suppressing activities of RB protein. Here, we show that clones of RB-reconstituted retinoblastoma cells can be isolated that stably express apparently normal RB protein for at least 20 months of continuous culture. These clones were indistinguishable from nonreconstituted cells by multiple parameters including morphology, growth rate, and cell cycle distribution. Despite similar phenotypes in culture, clones with stable RB expression were uniformly nontumorigenic in nude mice, whereas those that lost such expression regained their tumorigenic properties. These results indicate that the tumorigenicity of these cells is entirely determined by the presence or absence of exogenous RB protein expression and that suppression of tumorigenicity is distinct from inhibition of cellular growth in culture.


Inducible transformation of cells from transgenic mice expressing SV40 under lac operon control. If it were possible to clone in vitro cells of any type, at any stage of differentiation, from an extensively characterized animal such as the mouse, many areas of cell biology would benefit. Indeed, it would be even more helpful if these cells could subsequently be restored to their normal in vivo phenotype whenever required. Here, we describe a step on the pathway to such an idealized "clonable" mouse. In principle, it seeks to link a "universal" transforming agent to a regulatory system that is relatively simple, yet quite foreign to the mouse. A plasmid containing the bacterial lac operator/promoter region linked to the SV40 large T antigen and a vector containing the lac repressor that can be expressed in mammalian cells were coinjected into fertilized mouse oocytes utilizing the standard techniques for generating transgenic mice. Two progeny were obtained that express large T antigen in the presence, but not the absence, of the nonmetabolizable lac inducer, isopropyl-beta-thio-D-galactoside. This report characterizes fibroblast cell lines established from these transgenics that are readily transformed in vitro with isopropyl-beta-thio-D-galactoside. A significant proportion of the cells are restored to their "normal" (nontransformed phenotype) when isopropyl-beta-thio-D-galactoside is removed.


Erks: their fifteen minutes has arrived. In conclusion, a multigene family (ERK) encoding protein kinases that have the capacity to convert tyrosine kinase signals to serine/threonine phosphorylation signals has been identified in animal and yeast cells. Protein kinases from this family have been shown to be phosphorylated on tyrosine and threonine in response to mitogens, as well as to have the capacity to autophosphorylate on these amino acid residues. In contrast, they apparently phosphorylate exogenous substrates on serine and/or threonine. Studies with cultured cells, Xenopus, and sea star oocytes have furthered our understanding of possible functions of Erks in vivo. These enzymes respond immediately to extracellular signals and are involved in G0-G1 transition (cultured cells), as well as in the M phase of oocyte maturation (Xenopus and sea star oocytes). Their usage of MAPs as substrates in vivo suggests a possible role of Erks in microtubule reorganization. ERK-encoded protein kinases use c-Jun, EGF receptor, and Raf-1 as potential substrates and can also reactivate dephosphorylated S6 kinase in vitro. Taken together, these data suggest that these enzymes play an important role in relaying the mitogenic signal by phosphorylating down-stream kinases and specific transcriptional factors, as well as having possible feedback function in the process of signal transduction. The results from the study of the yeast enzymes are pertinent to Erk activation in cells with nonmitogenic responses described above. In such cases, Erk protein kinases may act directly or indirectly on cyclins to arrest division and permit differentiation. The pathways influenced by ERK-like gene products in animal and yeast cells suggest that, depending on the downstream targets of substrates, transcriptional changes in a particular cell may occur to drive the cell cycle or, alternatively, withdrawal from the cell cycle may lead to specific differentiation events.(ABSTRACT TRUNCATED AT 250 WORDS)


Discordant transforming growth factor beta 1 RNA and protein localization during chemical carcinogenesis of the skin. Transforming growth factor beta (TGF-beta) inhibits proliferation of normal keratinocytes, and this response is retained, to variable extents, in benign tumors of the skin (S. Haddow, D. J. Fowlis, K. Parkinson, R. J. Akhurst, and A. Balmain, Oncogene, 6: 1465-1470, 1991). To investigate the profile of TGF-beta biosynthesis during various stages of chemical carcinogenesis of the skin, we used a combination of ribonuclease protection assay, in situ hybridization with gene-specific probes for TGF-beta 1, -beta 2, and -beta 3, and immunohistochemistry with isoform-specific antibodies against TGF-beta 1. Following 12-O-tetradecanoylphorbol-13-acetate treatment of adult mouse skin, there was a rapid induction of TGF-beta 1 protein. Intracellular TGF-beta 1 protein was localized to suprabasal keratinocytes, and the extracellular form was localized predominantly to the dermis. Despite ubiquitous induction of TGF-beta 1 protein by 12-O-tetradecanoylphorbol-13-acetate in various mouse strains, we noted strain-specific differences in the quantitative induction of TGF-beta 1 RNA. Papillomas and carcinomas induced in vivo had elevated levels of TGF-beta 1 RNA within the basal keratinocyte compartment but did not contain significant levels of TGF-beta 1 protein within the tumor. We postulate that the tumor evades TGF-beta 1-controlled negative growth regulation by altered translational and/or posttranslational processing mechanisms of this growth factor. Levels of TGF-beta 2 and -beta 3 RNA were not elevated at any stage of chemical carcinogenesis of the skin.


Evoked potential and psychophysical analysis of Fourier and non-Fourier motion mechanisms. Some visual stimuli produce a strong percept of motion, even though they fail to excite motion detectors based on Fourier energy or cross correlation. Models which suffice to explain the motion percept in these non-Fourier motion (NFM) stimuli include linear spatiotemporal filtering, followed by rectification, followed by standard motion analysis (Chubb & Sperling 1988). We used the human "motion-onset" evoked potential, which has been assigned to area 17 on the basis of work in the macaque (van Dijk et al., 1986; van Dijk & Spekreijse, 1989), to investigate the neural substrate of the processing stages postulated in the above models. Motion-onset VEPs elicited by FM and NFM matched for spatial and temporal characteristics were indistinguishable in temporal characteristics and scalp topography at a transverse chain of electrodes. Addition of textural cues (granularity and higher-order form) did not influence the response dynamics or scalp topography of NFM responses. However, comparison of responses to NFM stimuli and related stimuli without coherent motion but similar spatial and temporal properties showed that the motion-onset responses were distinct from responses to the onset of fixed flicker-defined contours not undergoing coherent motion. We discuss the implications of these results for computational models of motion analysis.


Visual receptor cycle in normal and period mutant Drosophila: microspectrophotometry, electrophysiology, and ultrastructural morphometry. Visual pigment, sensitivity, and rhabdomere size were measured throughout a 12-h light/12-h dark cycle in Drosophila. Visual pigment and sensitivity were measured during subsequent constant darkness [dark/dark (D/D)]. MSP (microspectrophotometry) and the ERG (electroretinogram) revealed a cycling of visual pigment and sensitivity, respectively. A visual pigment decrease of 40% was noted at 4 h after light onset that recovered 2-4 h later in white-eyed (otherwise wild-type, w per+) flies. The ERG sensitivity [in w per+ flies in light/dark (L/D)] decreased by 75% at 4 h after light onset, more than expected if mediated by visual pigment (MSP) changes alone. ERG sensitivity begins decreasing 8 h before light onset while decreases in visual pigment begin 2 h after light onset. These cycles continue in constant darkness (D/D), suggesting a circadian rhythm. White-eyed period (per) mutants show similar cycles of visual pigment level and sensitivity in L/D; per's alterations, if any on the D/D cycles were subtle. The cross-sectional areas of rhabdomeres in w per+ were measured using electron micrographic (EM) morphometry. Area changed little through the L/D cycle.


Microspectrophotometric determinations of rod visual pigments in some adult and larval Australian amphibians. Visual pigments from the red rods of adults of eight species of Australian anuran amphibians, from a variety of habitats, were analyzed by microspectrophotometry. The lambda max in all cases fell between 502 nm and 506 nm, and the absorption spectra were well fitted by an A1-based visual pigment template curve. Red rod pigments were also analyzed for a number of tadpoles. In some cases the data were best fitted with an A1-based visual pigment template, in other cases with an A2-based template, and finally some tadpoles appeared to have mixtures of the two pigments.


Binocular rivalry suppression disrupts recovery from motion adaptation. The motion aftereffect (MAE) lasts longer when the test period does not immediately follow adaptation, a phenomenon called storage. Does storage of the MAE occur if the test target is present but rendered phenomenally invisible owing to the presence of a rival target presented to the other eye during the storage period? Our experiment addressed this question. Following adaptation to a drifting grating, an intervening period preceded testing with a stationary grating. During this period, the adapted eye either viewed the test target immediately or was occluded, and the unadapted eye either viewed a high-contrast rival target or was occluded. Thus four conditions were employed. The duration of the residual MAE was found to be longer for the rivalry condition (grating and rival target viewed) than for the normal MAE condition (grating viewed), and comparable to that in the stored MAE condition (both eyes occluded). Thus, the MAE is stored when the test target is rendered invisible due to binocular rivalry, indicating that a suppressed target is ineffective at promoting decay of the MAE. So while suppression does not prevent information about the adapting grating from reaching the site of generation of the MAE (Lehmkuhle & Fox, 1975), it can prevent information about the test target from reaching the site of the stored MAE. Current models attribute the MAE to reduced responsiveness of direction-selective cortical neurons (Sutherland, 1961; Barlow & Hill, 1963). Thus, storage should reflect a differential return of these adapted cells to preadapted response levels, dependent on postadaptation stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)


A breakdown of the blood-brain barrier is associated with optic nerve regeneration in the frog. We have examined the integrity of the blood-brain barrier during optic nerve regeneration in the frog Litoria (Hyla) moorei using rhodamine B-labeled bovine serum albumin (RBA). A transient localized breakdown of the blood-brain barrier was observed between 1 and 5 weeks after extracranial optic nerve crush. The zone of breakdown progressed along the experimental optic nerve, ascended the opposite optic tract, and swept rostro-caudally across the tectum contralateral to the crushed nerve. By 7 weeks, the blood-brain barrier was once again intact along the length of the optic pathway. In a concurrent series of frogs, regenerating optic axons were visualized by anterograde transport of horseradish peroxidase (HRP). At each stage examined, the region reached by the front of regenerating axons corresponded to that in which the blood-brain barrier had been shown to break down. In contrast to the results after nerve crush, the blood-brain barrier remained intact along the length of the optic pathway following optic nerve ligation to prevent regeneration. We conclude that the breakdown of the blood-brain barrier which occurs during optic nerve regeneration in the frog is triggered by the regenerating axons.


Visual deprivation fails to reduce calbindin 28kD or GABA immunoreactivity in the rhesus monkey superior colliculus. Antibody labeling of the calcium-binding protein calbindin 28kD (CaBP) and gamma-aminobutyric acid (GABA) is altered by short-term monocular deprivation in the lateral geniculate nucleus and visual cortex of adult primates. It is not known whether these alterations occur in other subcortical visual structures. We therefore have examined antibody labeling to CaBP and GABA in the superior colliculus (SC) of visually deprived Rhesus monkeys. One group was monocularly enucleated as adults. The other monkeys experienced different types of monocular and binocular deprivation from birth, including occlusion of one eye, and/or surgically induced aphakia, optically corrected with extended-wear contact lenses, or an intraocular lens implant. Some of these monkeys also had one eye enucleated prior to perfusion. In the SC of normal monkeys, CaBP-immunoreactive neurons formed three laminar tiers within SC, one within the zonal layer (ZL) and upper superficial gray layer (SGL), another bridging the optic and intermediate gray layers, and a third within the deep gray layer. CaBP neurons within the upper tier had small pyriform or stellate morphologies while those in the deeper tiers were slightly larger neurons, most with a stellate morphology. GABA-immunoreactive neurons were densely distributed within the SGL and more sparsely distributed within the deeper layers. These cells were mostly small neurons with horizontal, pyriform, or stellate morphologies. Neither monocular enucleation nor occlusion nor aphakia combined with continuous occlusion of the fellow eye produced any visible reduction in antibody labeling in cells or neuropil within the SC. Full-field measures of labeling intensity (optical density) within the ZL and upper SGL revealed no consistent differences between the SC contralateral or ipsilateral to the affected eye in either CaBP- or GABA-labeled sections. Measures of the optical density, number, and size of labeled neurons also showed no consistent effects of enucleation and/or occlusion. We therefore conclude that the retino-geniculostriate and retino-collicular systems differ in their response to deprivation which is likely due to the significant overlap of retinal axons from the two eyes that occurs in the SC of the Rhesus monkey.


Packing geometry of human cone photoreceptors: variation with eccentricity and evidence for local anisotropy. Disorder in the packing geometry of the human cone mosaic is believed to help alleviate spatial aliasing effects. To characterize cone packing geometry, we gathered positions of cone inner segments at seven locations along four primary and two oblique meridians in an adult human retina. We generated statistical descriptors based on the distribution of distances and angles to Voronoi neighbors. Parameters of a compressed-jittered model were fit to the actual mosaic. Local anisotropies were investigated using correlograms. We find that (1) median distance between Voronoi neighbors increases with eccentricity, but the minimum distance is constant (6-8 microns) across peripheral retina; (2) the cone mosaic is least compressed and jittered at the edge of the foveal rod-free zone; (3) disorder in the foveal center resembles that described by Pum et al. (1990); (4) cone spacing is 10-15% less in one direction than in the orthogonal direction; and (5) cone spacing is greater in the radial direction (along meridians) than in the tangential direction (along lines of isoeccentricity). The nearly constant minimum distance implies that high spatial frequencies may be sampled even in peripheral retina. Local anisotropy of the cone mosaic is discussed in relation to the growth of the primate retina during development and to the orientation biases of retinal ganglion cells.


Normalization of cell responses in cat striate cortex. Simple cells in the striate cortex have been depicted as half-wave-rectified linear operators. Complex cells have been depicted as energy mechanisms, constructed from the squared sum of the outputs of quadrature pairs of linear operators. However, the linear/energy model falls short of a complete explanation of striate cell responses. In this paper, a modified version of the linear/energy model is presented in which striate cells mutually inhibit one another, effectively normalizing their responses with respect to stimulus contrast. This paper reviews experimental measurements of striate cell responses, and shows that the new model explains a significantly larger body of physiological data.


Functional binocular vision is not dependent on visual experience in the praying mantis. In vertebrates, it has been shown that binocular visual experience is necessary to develop normal spatial vision. We have investigated whether this is also true for an invertebrate, the praying mantis. The praying mantis is a predatory insect in which prey localization involves the use of binocular disparities. We raised mantids which had one eye occluded throughout development and tested monocular visual fixation and binocular distance estimation in the adult animals. The results revealed that both fixation and prey catching behavior were normally functional in the monocularly reared animals. Thus we conclude that, in mantids, binocular vision is based on a fixed mode of development.


NADPH-diaphorase reactivity in the ventral and dorsal lateral geniculate nuclei of rats. The present study describes the patterns of NADPH-diaphorase reactivity in the ventral and dorsal lateral geniculate nuclei of rats. In the ventral lateral geniculate nucleus, two distinct populations of NADPH-diaphorase reactive cells are apparent. One population is deeply stained, generally larger in somal size and located in the more superficial or dorsolateral regions of the nucleus. The second population of reactive cells in the nucleus is lightly labeled, small in somal size, and found in deeper or more ventromedial regions of the nucleus. Double labeling with an antibody to GABA revealed that neither cell class is GABAergic. In the dorsal lateral geniculate nucleus, reactivity is apparent in lightly labeled small cells only, most of which are GABA immunoreactive also. The NADPH-diaphorase reactive cells, however, form only a small proportion of the total population of GABAergic cells in the nucleus. The striking feature of the NADPH-diaphorase reactive cells in the dorsal lateral geniculate nucleus is their spatial distribution. Most cells are located in the more superficial or dorsolateral areas: very few are apparent in deeper or more ventromedial areas of the nucleus. This distribution closely parallels the location of the outer "shell" region of the nucleus (see Reese, 1988), which receives most of its afferents from the smaller class II and III ganglion cells of the retina and from the superior colliculus.


The assessment of maritally violent men on the California Psychological Inventory. This study assessed 87 maritally violent men (MV), 42 maritally nonviolent, maritally discordant men (NVD), and 48 maritally nonviolent, maritally satisfied men (NVS) on the California Psychological Inventory (CPI), a test of the normal personality. A MANOVA and subsequent range tests indicated that the NVD and NVS groups had significantly higher scores than the MV group on 10 of the 18 subscales: Responsibility, Socialization, Self-Control, Tolerance, Achievement via Conformance, Achievement via Independence, Good Impression, Intellectual Efficiency, and Psychological Mindedness. A discriminant analysis contrasting the MV group with the combined NVD and NVS group correctly classified 68% of the subjects and accounted for 20.94% of the variance between groups. Along with previous findings, the data indicated that maritally violent males exhibit different personality characteristics than maritally nonviolent men in three general areas: intimacy, impulsivity, and problem-solving skills. Many of these problem areas were significantly correlated with childhood violence experiences.


Patterns of socially desirable responding among perpetrators and victims of wife assault. Wife assaulters attending a treatment group and women who had just exited an abusive relationship were asked to report on the extent of physical violence and emotional abuse in their relationship. Measures of socially desirable responding (SDR) were administered to both groups. Wife assaulters' self-reports of physical abuse correlated negatively with one SDR measure (the Balanced Inventory of Desirable Responding) but not another (the Marlowe-Crowne Social Desirability Scale); emotional abuse correlated negatively with both measures. Although physical abuse was primarily related to impression management, psychological abuse was affected by both impression management and self-deception aspects of SDR. Wife assaulters' reports of their own anger also correlated negatively with SDR. Both self-deception and impression management appear to contribute to underreporting of anger. Finally, abuse victims' reports of both physical and emotional abuse were unrelated to SDR.


Dating violence, social learning theory, and gender: a multivariate analysis. The study of violence between dating partners is a logical extension of interest in marital violence. However, little of this research tests explanations of intimate violence using multivariate techniques, and only recently have such tests occurred within a theoretical framework. Drawing on a recent social learning model of courtship violence (Riggs & O'Leary, 1989), this paper empirically examines constructs hypothesized to be predictive of the use of dating violence and investigates possible gender differences in the underlying causal structure of such violence. Logit analysis indicates that parent-child violence, drug use, and knowledge of use of dating violence by others predict the use of courtship violence by females. Belief that violence between intimates is justifiable, drug use, and parental divorce are related to perpetration of dating aggression by males. Explanations for these results and the importance of a multivariate approach to the problem are discussed.


A structural model examining the relationship between physical child abuse, sexual victimization, and marijuana/hashish use in delinquent youth: a longitudinal study. A structural model of the relationships among physical abuse and sexual victimization experiences, marijuana/hashish use (measured by self-report and urine test data) and self-reported delinquent behavior (theft crimes, index offenses, crimes against persons, drug sales and total delinquency) over time was tested in a longitudinal study of juvenile detainees. The hypothesized model was supported by the data. Theoretical, research and policy implications of the results are drawn.


The nuts and bolts of implementing large-scale longitudinal studies. Studies that assess large numbers of subjects for longitudinal research, for epidemiological purposes, or for the evaluation of prevention and intervention efforts, are very costly and should be undertaken with the greatest care to ensure their success. The success of a study, apart from its scientific merit, depends largely on the ability of the researcher to plan and set up a smoothly running operation. However, the skills required for such a task are often not acquired in academic training, nor do scientific journals abound with information on the practical aspects of running a large study. This paper summarizes the experience gained in executing a longitudinal study and covers aspects of planning, hiring of staff, training and supervision of interviewers, data collection and data entry and management. The importance of the use of the computer as a management tool is stressed.


Recruiting nonviolent men as control subjects for research on marital violence: how easily can it be done? Data regarding the prevalence of violence among subjects recruited for nonviolent control groups in five studies are presented. These studies examined subjects recruited using a variety of methods, including couples seeking marital therapy and couples recruited from newspaper advertisements. Across the studies, it appears that up to one-third of maritally nondistressed couples and one-half of maritally distressed couples report that husband violence has occurred in their relationship. The implications of these data for the recruitment of control groups in future studies of marital violence are discussed, and questions regarding the ease of recruiting nonviolent couples for control groups are raised.


Monomer units for the beta-bend ribbon structure: MeAib peptides. A conformational analysis in CDCl3 solution by using i.r. absorption and 1H nuclear magnetic resonance spectroscopy was performed on the fully blocked dipeptides Z-MeAib-Aib-NHMe, Z-MeAib-L-Ala-NHMe, Z-Aib-MeAib-NHMe, and Z-L-Ala-MeAib-NHMe, representing repeating units of the beta-bend ribbon spiral (an approximate 3(10)-helix, with an intramolecular H-bonding donor every two residues), where Z represents benzyloxycarbonyl, MeAib alpha-methylaminoisobutyric acid, and NHMe methylamino. The molecular and crystal structures of the first three compounds were also assessed by X-ray diffraction. While the -MeAib-Aib-, -MeAib-L-Ala-, and -Aib-MeAib- sequences give stable beta-bend structures, the preferred conformation of the -L-Ala-MeAib- sequence is open. These results indicate that the MeAib residue is a good beta-bend promoter, but less efficient than its unmethylated counterpart at position i + 2.


Modes of binding of alpha (1-2) linked manno-oligosaccharides to concanavalin A. Three-dimensional structures of the complexes of concanavalin A (ConA) with alpha(1-2) linked mannobiose, triose and tetraose have been generated with the X-ray crystal structure data on native ConA using the CCEM (contact criteria and energy minimization) method. All the constituting mannose residues of the oligosaccharide can reach the primary binding site of ConA (where methyl-alpha-D-mannopyranose binds). However, in all the energetically favoured complexes, either the non-reducing end or middle mannose residues of the oligosaccharide occupy the primary binding site. The middle mannose residues have marginally higher preference over the non-reducing end residue. The sugar binding site of ConA is extended and accommodates at least three alpha(1-2) linked mannose residues. Based on the present calculations two mechanisms have been proposed for the binding of alpha(1-2) linked mannotriose and tetraose to ConA.


The contribution of halogen atoms to protein-ligand interactions. The three-dimensional structure of para-fluoro-D-phenylalanine (PFF) in its complex with the zinc protease carboxypeptidase A (CPA) has been determined at 2.0 A resolution by X-ray crystallographic methods. The structure reveals that the para-fluorobenzyl side chain of the inhibitor is buried in the S'1 hydrophobic pocket of the enzyme. Intriguingly, this ligand molecule inhibits CPA better than its amino acid analogues D-phenylalanine (D-Phe) and D-tyrosine (D-Tyr) by factors of 4 and 5, respectively. Moreover, the para-fluoro derivative is a better inhibitor than para-chloro- or para-bromo-D-phenylalanine by nearly a factor of 50. This result is consistent with binding enhancements realized in other protein complexes involving halogenated ligand molecules, regardless of whether the carbon-halogen group of the ligand makes specific polar interactions or non-specific hydrophobic interactions with its protein host. In the CPA-PFF complex, the fluorine atom of PFF does not make any direct polar contact with the enzyme, and the contact surface area of the protein-ligand interface is only slightly greater, although more hydrophobic, than that of D-Phe and D-Tyr. Therefore, we conclude that the slight binding enhancement measured for PFF relative to D-Phe and D-Tyr arises predominantly from increasing the hydrophobic character of the protein-ligand interface, and not solely from increasing the degree of protein-ligand contact.


Chemical modification of arginyl residues in silk fibroin: 1. Reaction of 1,2-cyclohexanedione in borate buffer. Chemical modifications of silk fibroin were attempted in order to add new properties and functions to silk fibroin. The arginyl residue in solubilized silk fibroin was chemically modified with the reaction of 1,2-cyclohexanedione in borate buffer. FT-i.r. and c.d. spectra of the silk fibroin before and after the modification indicated that the fraction of random coil conformation increased with the modification. The chemical stability of the modified silk fibroin membrane was investigated in vitro with phosphate buffer. The modified arginyl residue in the membrane was considerably regenerated with the treatment in phosphate buffer.


Interaction of two fragments of cibacron blue F3GA having anthraquinone chromophores with basic polypeptides. The interaction of two anthraquinone dyes, bromaminic acid 1-amino-4-bromo anthraquinone-2-sulphonic acid (BA) and 1-amino-4-(4'-aminophenylamino)-anthraquinone-2,3'-disulphonic acid (ASSO), with poly-L-lysine (PLL), poly-L-ornithine (PLO) and poly-L-arginine (PLA) has been studied. The flexibility of the side chain (chain length) and the geometry of the charge centre (tetrahedral/planar) of the polypeptide were found to affect the interaction. Further, both dyes induce ordered conformation (alpha or beta) for the three polypeptides, but the final conformation was found to depend on the polypeptide as well as the number of anionic sites on the ligand. While the bidentate ASSO-polypeptide complexes acquire beta-conformation, the monodentate BA distinguished between the three substrates and induces beta-conformation only for PLO.


Chemical modification of proteins under high pressure. Preparation of ferrocene-attached bovine serum albumin and glucose oxidase. High hydraulic pressure was used for denaturing proteins during chemical modifications. Bovine serum albumin and glucose oxidase were selected as the first targets of this unique technique and the ferrocene group was introduced into them, to obtain a macromolecular electron mediator and a self-electron-mediating oxidase, respectively. The result was compared with those obtained under non-denaturing conditions and under urea-denatured conditions. As for the number of ferrocene group linked to the protein, the pressure denaturation is superior to chemical denaturants, where ferrocenecarboxyaldehyde was used as the modifier. In both proteins the ferrocene group seemed to be introduced mainly inside the molecules with the pressure method, as the native conformation of the protein was restored when the high pressure was removed.


Studies on the function mechanism of a Formosan grey mullet protamine-mugiline beta M6: interaction of the M6 and M6 fragments with DNA. The interaction of three peptide segments of one component of Formosan grey mullet protamine (mugiline beta M6), obtained by chemical and enzymatic cleavage, with DNA was studied by spectroscopic measurement, thermal denaturation and circular dichroism. The data obtained were then compared with those of whole M6 and other fish protamines such as salmine of salmon and clupeine of herring. M6-B-I, which lacks C-terminal 11 amino acids in M6, showed significantly different properties. It showed remarkably high DNA aggregating ability which was due to a conformational change of DNA from B to A form. The conformational change of DNA induced by the binding of M6-B-I was reproduced by the carboxypeptidase B digestion of DNA-M6 complex. From these results, the arginine-rich, C-terminal domain of the M6 molecule was estimated to be essential for natural DNA binding.


Single crystals of alpha-chitin.

Single crystals of alpha-chitin. Single crystals of alpha-chitin were grown by the addition of precipitants to dilute solutions of low molecular weight chitin fractions dissolved in aqueous LiSCN. At temperatures around 200 degrees C, bundles of thin needle-shaped crystals were obtained. Each of these needles was an alpha-chitin single crystal, characterized by a spot electron diffraction pattern which could be indexed along the hk0 reciprocal net corresponding to the Minke and Blackwell unit cell [a = 0.474 nm, b = 1.88 nm, c (fibre axis) = 1.032 nm, space group P2(1)2(1)2(1)]. In a crystal, the a* parameter was along the crystal axis and the b* perpendicular to it.


Studies on chitosan: 4. Lysozymic hydrolysis of partially N-acetylated chitosans. The lysozymic digestibility of partially N-acetylated chitosans was studied by measuring the reducing sugars produced and the molecular weights of their hydrolysates. Moderately N-deacetylated chitosans (MDC), obtained by N-deacetylation of chitin under heterogeneous conditions, were about four times more digestible at an early stage than partially N-acetylated chitosans (PAC-H) with similar acetyl content, prepared by N-acetylation of highly N-deacetylated chitosans under homogeneous conditions. The molecular weights of the hydrolysates of MDC decreased rapidly but gradually reached a constant value in contrast to the behaviour of PAC-H. The Km was 0.14 mM for 30% N-acetylated MDC and 0.12 mM for 65% N-acetylated PAC-H although the degree of N-acetylation of the latter was twice as much as the former. These differences were due to the different distribution patterns of N-acetyl groups in two types of the chitosans. MDC with 20-30% acetyl content have the sequences of more than three N-acetyl-D-glucosamine residues but PAC-H with about 30% acetyl content are random-type copolymers of N-acetyl-D-glucosamine and D-glucosamine units. PAC-H with more than 50% acetyl content have the sequences of more than three N-acetyl-D-glucosamine residues.