Childhood leukaemia following the Chernobyl accident: the European Childhood Leukaemia-Lymphoma Incidence Study (ECLIS). The objective of the European Childhood Leukaemia-Lymphoma Incidence Study (ECLIS) is to investigate trends in incidence rates of childhood leukaemia and lymphoma in Europe, in relation to the exposure to radiation which resulted from the accident at the Chernobyl nuclear power plant in April 1986. In this first report, the incidence of leukaemia in children aged 0-14 is presented from cancer registries in 20 European countries for the period 1980-1988. Risk of leukaemia in 1987-1988 (8-32 months post-accident) relative to that before 1986, is compared with estimated average dose of radiation received by the population in 30 geographic areas. The observed changes in incidence do not relate to exposure. The period of follow-up is so far rather brief, and the study is planned to continue for at least 10 years.


In vitro and in vivo antitumoral activity of alkylphosphonates. Hexadecylphosphocholine is a new antitumour agent with a highly selective activity in chemically induced mammary tumours. It was suggested, that hexadecylphosphocholine is a pro-drug, cleavable by phospholipases C and/or D, creating hexadecanol or hexadecylphosphate as the active principle. To test this hypothesis, the antineoplastic activity of three alkylphosphonates, cleavable either by phospholipase C or D, are compared with those of the parent compound, hexadecylphosphocholine. Cell culture experiments, in which radiolabelled alkylphosphonates were incubated with a neoplastic cell line, showed no metabolism even after 3 days of incubation. In in vivo experiments with dimethylbenzanthracene-induced rat mammary carcinomas, all three alkylphosphonates showed antineoplastic activity, although none of them reached the high activity of hexadecylphosphocholine. These results indicate that the antitumoral activity of alkylphosphocholines and alkyl lysophosphatidylcholines is due to direct toxicity and not dependent on metabolism by phospholipases C or D or related enzymes.


Influence of aw value and storage temperature on the multiplication and enterotoxin formation of staphylococci in dry-cured raw hams. Growth and enterotoxin A production in dry-cured raw ham, with different aw and pH values, was investigated for Staphylococcus aureus strain FRJ-100 over a temperature range of 20-35 degrees C when stored for up to 7 days. Enterotoxin production took place at all temperatures tested in the meat of ham that was cured, but not dried (aw 0.95). In dry-cured raw ham with an aw-value of about 0.92, and stored at 20 degrees C, no enterotoxin production was detectable within the 7-day storage period, whereas enterotoxin was produced at the higher temperatures. In ham with an aw of about 0.89, enterotoxin was produced only when stored for 7 days at 35 degrees C. Critical points for S. aureus multiplication and enterotoxin-formation during production and storage of dry-cured raw ham are discussed.


Detection of Clostridium botulinum in natural sweetening. Various sugar products were examined for contamination with C. botulinum spores. Type A, B and C spores were detected in three of 56 samples of sugar for apiculture, which may attest the significance of bee-feed as a source of contamination of honey. The heavy contamination of honey with C. botulinum spores sometimes encountered, however, can not be explained unless some other factors, e.g., that allowing germination and multiplication of the spores somewhere during honey production, are found. Type A spores were detected in some samples of raw sugar and molasses and also in two of 41 samples of brown sugar lump, but not in refined sugar or other various samples taken at a sugar factory or in sugar cane left on the field in Okinawa. The fact that some natural sweetenings are contaminated with C. botulinum spores, even in low concentrations, may be food-hygienically important.


Fungi causing thread mould spoilage of vacuum packaged Cheddar cheese during maturation. Thread mould is a defect which occurs sporadically in maturing vacuum packaged Cheddar cheese, caused by the growth of fungi in folds and wrinkles of the plastic film in which the cheese is packaged. Fungi were isolated and identified from 110 Cheddar cheese blocks exhibiting typical thread mould defects. The major causative species were found to be Cladosporium cladosporioides, Penicillium commune, C. herbarum, P. glabrum and a Phoma species. Yeasts were also frequently isolated from the cheese, the majority belonging to the genus Candida. Fungal species which can cause thread mould defects were also found in the cheese factory environment, on cheesemaking equipment, in air, and in curd and whey, providing a wide range of potential sources of contamination.


Variation in growth kinetics and phenotype of Aeromonas spp. from clinical, meat processing and fleshfood sources. Sixty-four strains of motile aeromonads (A. hydrophila, A. sobria and A. caviae), isolated from clinical meat processing and ready-to-eat fleshfood sources, and the A. hydrophila type strain were tested with respect to their growth kinetics at 4 degrees C and 37 degrees C, and the reported indicators of pathogenicity: autoagglutination and haemolysis (tested using a CAMP reaction). Between the species, A. caviae grew the fastest at 37 degrees C and had the highest percentage of strains not able to grow at 4 degrees C (after 200 h incubation). Within the species, food-derived strains of A. hydrophila were better adapted to growth at lower temperatures than those from clinical or meat processing sources. Clinical strains of A. hydrophila autoagglutinated more frequently than those from other sources, but not differences in CAMP reactions were noted. Aeromonas caviae and A. sobria isolates appeared to be homogeneous with respect to growth kinetics at the temperatures tested. A comparison of the growth kinetics of the A. hydrophila type strain and a food-derived A. hydrophila strain clearly reflected the latter's enhanced ability to grow at low temperatures.


Identification and characterization of two bacteriocin-producing strains of Lactococcus lactis isolated from vegetables. Isolated from mixed salad and fermented carrots, 123 strains of lactic acid bacteria were screened for bacteriocin production. Two strains, D53 and 23, identified as Lactococcus lactis by DNA-DNA hybridizations, produced heat stable bacteriocins which were resistant to trypsin and pepsin, but were inactivated by alpha-chymotrypsin and proteinase K. The bacteriocins were active from pH 2 to 9 and inhibited species of Listeria, Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Carnobacterium, Bacillus and Staphylococcus. Strain D53 produced bacteriocin at pH values of 4.5-8.0 and from 10 to 37 degrees C.


Incidence of Listeria monocytogenes in fresh foods in Barcelona (Spain). From September 1989 to March 1990, a study of Listeria spp. was carried out on 311 samples of raw foodstuffs from markets and other establishments in the city of Barcelona. These foodstuffs included vegetables (103 samples), minced meats from pork, beef and poultry (168 samples) and bivalve molluscs (40 samples). L. monocytogenes was isolated in 7.8% of the vegetable samples, 17.3% of the minced meats and in 7.5% of bivalve molluscs. The most frequent serovars were 1/2 and 4. Other species isolated were L. innocua, L. welschimeri and L. seeligeri.


The incidence of Listeria species in retail foods in Japan. Meat, fish and vegetable products obtained at retail shops in or around Tokyo were examined for Listeria contamination. Listeria spp. were isolated from 43 (56.6%) out of 76 samples of meat products. L. monocytogenes occurred in 26 (34%) of the samples, L. monocytogenes was isolated from 7 (6.1%) out of 114 samples of fish and fish products including 'ready-to-eat' foods. Listeria was not isolated from any of 21 samples of vegetable and vegetable product including 'ready-to-eat' foods investigated.


The ability of the ropy slime-producing lactic acid bacteria to form ropy colonies on different culture media and at different incubation temperatures and atmospheres. The ability of the ropy slime-producing lactic acid bacteria to form ropy colonies aerobically and anaerobically on APT, MRS, MRS-S and Rogosa SL agars at 15, 20 and 30 degrees C was tested. Wide variation was observed in the proportion of ropy colonies produced with different culture methods and different test periods. The percentage of viscous colonies was usually highest at 15 degrees C. No suitable uniform method for all strains was found; and the results varied according to the bacterial strain studied. For testing purposes, use of the method producing the highest proportion of ropy colonies for each bacterial strain is recommended.


Classification of ropy slime-producing lactic acid bacteria based on DNA-DNA homology, and identification of Lactobacillus sake and Leuconostoc amelibiosum as dominant spoilage organisms in meat products. The classification of lactic acid bacteria able to cause ropy slime on vacuum-packed cooked meat products was carried out based on DNA-DNA homology. The ropy slime-producing lactobacilli were identified as strains of Lactobacillus sake and the ropy slime-producing leuconostocs, such as Leuconostoc amelibiosum and Leuconostoc mesenteroides.


Decontamination with lactic acid/sodium lactate buffer in combination with modified atmosphere packaging effects on the shelf life of fresh poultry. The effect of the treatment with various concentrations (2%, 5%, 7.5% and 10% w/v) of lactic acid/sodium lactate buffer (pH 3.0) combined with modified atmosphere packaging (MAP) (90% CO2/10% O2) on the shelf life and organoleptic quality of fresh chicken legs stored at 6 degrees C was investigated. The CO2 concentration of all samples packed in modified atmosphere (MA) decreased during the first 3 days of storage, followed by a gradual increase after the third day, while O2 showed a corresponding decrease. The buffering capacity of the buffer systems seem to be sufficient to maintain a low pH of the skin during storage. Legs treated with 2, 5, 7.5, and 10% (w/v) lactic acid/sodium lactate buffer (pH 3.0) combined with MAP have a shelf life at 6 degrees C of 14, 15, 16 and 17 days, respectively. The shelf life when the product was not treated with lactic acid was 1, 2.3 and 4 days shorter, respectively.


Incidence and coincidence of Listeria spp., motile aeromonads and Yersinia enterocolitica on ready-to-eat fleshfoods. A survey for the presence of Listeria spp., Yersinia enterocolitica and motile aeromonads in 203 samples of ready-to-eat fleshfoods purchased from retail outlets was conducted. Overall, 39.4%, 3.4% and 23.2% of samples were positive for the presence of Listeria spp., Y. enterocolitica and motile aeromonads respectively. Two factors have been identified as contributing to contamination of fleshfoods by these cold-tolerant bacteria. These are (i) the method of sale; delicatessen-bought foods were notably more contaminated than similar products bought pre-packaged, and (ii) the method of preservation. For motile aeromonads fermented foods were the least contaminated, whereas smoked and cooked products had similar incidence rates. For L. monocytogenes, significantly more (41.9%) smoked products were contaminated than fleshfoods preserved by other methods. For Y. enterocolitica, only cooked products were contaminated. In the case of cooked fleshfoods it must be assumed that most contamination occurs post-cooking and that contamination rates are increased by poor food handling procedures. Of the three possible pairwise combinations of these organisms, the coincidence of Y. enterocolitica and motile aeromonads was the only one that differed significantly from a random distribution (P less than 0.001), indicating that fleshfoods contaminated with Y. enterocolitica are probably also contaminated by motile aeromonads.


Is any strain of Listeria monocytogenes detected in food a health risk? Listeria spp. have been isolated from various food items. This fact does not mean in any case a true health risk. A balanced appraisal should be based on quantitative as well as qualitative aspects. Actually, there is still an open debate whether a limited number of Listeria has to be tolerated at least in certain food items. In addition, the pathogenic potency of an isolate may be put into account. Pathogenicity of various Listeria spp. definitely varies. Most Listeria spp., except Listeria monocytogenes, can be regarded as harmless to man. Also, not all strains of L. monocytogenes are pathogenic: rough variants possess only reduced virulence; non-hemolytic mutants have completely lost their pathogenic potency. Furthermore, several other virulence factors may be lost under natural conditions, so that among the majority of hemolytic, pathogenic isolates there may be others which are non-pathogenic or of low virulence only. Unfortunately, these strains actually cannot be recognized and characterized by common laboratory tests, so that animal pathogenicity seems to be the only way to get a final conclusion on the health risk of an isolate of L. monocytogenes from any food. The problem raised by this is which animal test is able to predict a true health risk either for normal hosts or for immunocompromised patients?


Characterization and partial purification of a bacteriocin produced by Leuconostoc carnosum LA44A. Twenty Leuconostoc strains isolated from vacuum packaged Vienna-type sausages were screened for antagonistic activity against various Gram-positive organisms (including Listeria spp.). One of the three strains exhibiting inhibitory activity was chosen for further investigation. This strain was identified as Leuc. carnosum and the inhibitory substance produced was named carnosin. Carnosin was inactivated by trypsin but not by catalase or other non-proteolytic enzymes tested. Carnosin retained activity after heating at 100 degrees C for 20 min, whereas heating at 121 degrees C for 15 min resulted in complete loss of activity. Carnosin was active at pH values ranging from 2 to 9. Carnosin activity was not detectable until cells were in the late log-phase of growth. At low temperatures (4 degrees C), higher cell densities were required before carnosin activity could be detected. Carnosin was active against various lactic acid bacteria, Enterococcus faecalis and Enterococcus faecium and against Listeria spp. Difficulties in purification were reduced by growing Leuc. carnosum in a modified MRS medium, having 50% of the normal peptone concentration and no Tween or meat extract. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of partially purified carnosin indicated that it has a molecular mass between 2510 and 6000 Da. Yet, retention of activity after exhaustive dialysis suggested a molecular mass > 14kDa. It is hypothesized that carnosin forms large active complexes which can be dissociated to small (active) components.


Evaluation of the MPN, Anderson-Baird-Parker, Petrifilm E. coli and Fluorocult ECD method for enumeration of Escherichia coli in foods of animal origin. Commercially available beta-D-glucuronidase (GUR) based methods, Petrifilm E. coli (PEC) and Fluorocult ECD (FECD), and ISO standard MPN and Anderson-Baird-Parker (ABP) procedures were evaluated for routine enumeration of E. coli in naturally contaminated foods of animal origin. The methods concerned were classifiable in a sequence of best qualities for: production, MPN > ABP = PEC = FECD; costs, FECD > ABP = PEC > MPN; time per measurement, ABP = PEC = FECD > MPN; practical use, PEC > FECD > ABP > MPN; detection at low contamination, MPN > ABP = PEC > FECD. The ABP and PEC method appeared useful for routine counting of E. coli in raw meat, poultry and meat products, whereas the MPN procedure turned out to be more sensitive, however, impractical and considerably more expensive. The FECD method was inexpensive although suitable for the enumeration of E. coli at higher contamination level (> 50 cfu/g). The indole and MUG indicators both applied to demonstrate E. coli with the ABP or FECD method proved to be equal in specificity.


Heat-resistant fungi in the soil.

Heat-resistant fungi in the soil. The occurrence of heat-resistant fungi has been demonstrated in samples of soil from the Slovak Republic. The heat-resistant species isolated were Byssochlamys nivea, Dichotomomyces cejpii, Eupenicillium baarnense, Neosartorya fischeri, Talaromyces avellaneus, Tal. bacillisporus, Tal. emersonii, Tal. flavus, Tal. trachyspermus, Tal. wortmanii, Botryotrichum piluliferum, Gilmaniella humicola and Nodulisporium sp. Some of them were isolated for the first time from Czechoslovakian soil. For the various soil samples examined, the occurrence of heat-resistant fungi varied qualitatively and quantitatively. Further research is needed to identify conditions affecting the occurrence of heat-resistant fungi in soil.


Sakacin M, a bacteriocin-like substance from Lactobacillus sake 148. The antagonistic activity of Lactobacillus sake 148 was evaluated during its growth on complex broth media and in a semisynthetic defined medium (SDM) with various supplements. The antagonistic activity was a growth-associated property, being detected and quantified when L. sake 148 was grown at either 4, 8, 16, 25 or 32 degrees C. The concentrated culture supernatant of L. sake 148 was subjected to purification by lyophilization and gel filtration. The purification procedure resulted in a small increase in its specific activity (7-fold) and in a low recovery of the original inhibitory activity (8%). Gel filtration analysis of the partially purified activity on Sephadex G-50 revealed an apparent molecular weight of 4640. The partially purified antagonistic activity of L. sake 148 was destroyed by treatment with proteolytic enzymes. However, the antagonistic activity was resistant to heat, having D-values at 121, 135 and 150 degrees C of 23.8, 17.4 and 15.2 min, respectively.


Is thermotolerance correlated to heat-shock protein synthesis in Lactococcus lactis subsp. lactis? Exposure of Lactococcus lactis subsp. lactis cells to a heat shock at 40 degrees C for 30 min induces thermotolerance, the increased ability of bacterial cells to survive exposure to lethal temperature (52 degrees C for 25 min). This transient state of thermal resistance is accompanied, as in Escherichia coli, by the synthesis of a new set of specific proteins termed heat-shock proteins (Hsps). Pre-treatment of the bacterial cells by antibiotics (streptomycin, spiramycin, kanamycin and erythromycin) known to act on translation, induces the major Hsps synthesis but no thermal protection; conversely, puromycin and amino acid analogues treatments, known to produce abnormal and incomplete peptides, triggers the thermotolerance state without inducing significant Hsps synthesis. These results demonstrate that heat-shock response and induced thermotolerance are not tightly correlated phenomena in L. lactis subsp. lactis.


Mycological condition of maize products. Maize and maize-related products were investigated in a collaborative study for viable moulds and antigenic extracellular polysaccharides (EPS) produced by Aspergillus and Penicillium species. In addition, the samples were tested for the presence of aflatoxin B1. All maize products, with the exception of the heat processed products, contained viable moulds on an average of (log10 values) 3.3 +/- 0.7 colony-forming units per gram. In most samples a mixed mould flora was present. Species of the genus Fusarium were dominant, followed by Aspergillus, Eurotium and Penicillium. The mould colony count correlated positively with the presence of antigenic extracellular polysaccharides produced by species of Aspergillus and Penicillium. Gamma irradiation did not affect the detection of antigenic extracellular polysaccharides. Aflatoxin B1 was detected in two out of 35 samples; these contained 0.6 and 0.8 microgram/kg. From one of these aflatoxin B1-containing samples, Aspergillus flavus was isolated.


Differentiation of Listeria monocytogenes isolates by using plasmid profiling and multilocus enzyme electrophoresis. Three hundred and seven Listeria monocytogenes isolates from various origins (clinical sources, raw chicken, seafoods, dairy and meat products and processing environments) were screened for plasmids. The overall frequency of L. monocytogenes isolates containing plasmids was 77%. The highest percentages of plasmid positive isolates were found from meat (89%), chicken (81%) and dairy products (64%), while clinical isolates had the lowest plasmid percentage (28%). Seven sizes of plasmids (21, 24, 27, 35, 40, 47 and 52 MDa) were distinguished. All sizes were represented in the meat isolates, clinical isolates contained only two of the plasmid sizes, while several different sizes of plasmids were found in the isolates from other origins. Plasmid profiling divided the isolates into ten plasmid pattern types. Multilocus enzyme electrophoresis of 75 isolates demonstrated 12 distinctive multilocus genotypes (ETs) which clustered into two groups: cluster A including serotype 1 and 4 isolates, and isolates not typable by Difco antisera serotype 1 and 4, and cluster B containing only serotype 1 isolates. No relationship between ETs and plasmid profiles could be demonstrated.


Removal of Yersinia enterocolitica from fresh parsley by washing with acetic acid or vinegar. Different washing procedures and solutions were applied to fresh parsley and the effect on artificially contaminated Y. enterocolitica and the natural population of aerobic bacteria was determined. Dipping the parsley containing 10(7) Y. enterocolitica per gram into the 2% (v/v) acetic acid or 40% (v/v) vinegar solutions for 15 min exerted pronounced bactericidal effect against this organism. No viable aerobic bacteria were recovered after 30 min dip in 5% (v/v) acetic acid, whereas vinegar led to 3-6 log10 cycles decrease in the number of aerobic bacteria depending on vinegar concentration and holding time.


Impedance detection of Salmonella in processed animal protein and meat. The impedance technique showed a detection rate (95%) equal to that of conventional enrichment for raw meat contaminated with Salmonella. For processed animal protein samples impedance was less sensitive. A commercially available Easter and Gibson impedance medium used for the selective enrichment of salmonellae proved superior to the laboratory prepared equivalent for the detection of Salmonella in processed animal protein. The rate of false-positive results with the impedance technique was high.


Pros and cons of fluoxetine for the depressed cancer patient. When major depression develops in patients with cancer, specific antidepressant treatment should be initiated. Fluoxetine (Prozac) is one of several effective treatments for depression; it is currently the most frequently prescribed antidepressant in the United States. Unfortunately, the information from studies and reports regarding the use of fluoxetine in patients with cancer is limited. This article reviews the properties, drug interactions, and side-effect profiles of fluoxetine and the other antidepressants most relevant to the care of cancer patients. Also discussed are strategies for the prudent prescription of fluoxetine, the tricyclic antidepressants, and the psychostimulants in depressed cancer patients.


The human genome project and clinical medicine. Genetic research has already begun to pay clinical dividends, as investigators have successfully isolated disease genes, including those responsible for Duchenne muscular dystrophy, cystic fibrosis, and the fragile X syndrome. This last disorder appears to be associated with the progressive amplification of a short, repeated DNA sequence, a mechanism that may also occur at other cytogenetically fragile sites and in other genetic disorders or neoplasias. This article reviews genetic mapping techniques being used by the Human Genome Project, methods for identifying disease genes, and clinical applications. It also includes discussions of mutation detection, diagnosis, and gene therapy.


Adjuvant therapy of rectal cancer.

Adjuvant therapy of rectal cancer. The treatment of rectal cancer has gradually evolved based on our knowledge of the failure patterns. Although results with surgery alone are good in patients with disease limited to the bowel wall and with no lymph node spread, surgery alone is less effective for more advanced disease. Treatment strategies that combine pelvic radiation therapy and 5-fluorouracil-based chemotherapy regimens have significantly improved local recurrence rates and overall survival results for patients with disease extending through the bowel wall or those with positive lymph nodes. Recent studies suggest that methyl CCNU can be dropped from chemotherapy regimens without loss of efficacy. Newer strategies being investigated include modulations of 5-fluorouracil with agents such as leucovorin and levamisole, possibly in combination, and continuous infusions of 5-fluorouracil.


Isolation and characterization of cDNA encoding a spicule matrix protein in Hemicentrotus pulcherrimus micromeres. A cDNA clone, termed pHPSMC, was obtained from the Japanese sea urchin Hemicentrotus pulcherrimus, and it was found to be highly homologous in sequence to the spicule matrix protein cDNA of Strongylocentrotus purpuratus (Sucov et al., Dev. Biol. 120: 507-519, 1987). During early embryogenesis, mRNA complementary to pHPSMC appeared in gastrulae and remained at a similar level until the pluteus stage. In situ hybridization revealed that the mRNA was localized exclusively in primary mesenchyme cells in gastrulae. pHPSMC mRNA was detected in micromeres in vitro after 48 h of culture, but it was not found in blastomeres immediately after isolation. These features suggested that pHPSMC represents the spicule matrix protein cDNA cognate in Hemicentrotus pulcherrimus. In the derived polypeptide, we detected a domain containing a tandemly repeated 13-amino acid sequence as did Sucov et al. (1987). Unexpectedly, the sequence of the repeated element was completely different from that originally reported for Strongylocentrotus purpuratus, but it was very similar to the corrected sequence that appeared recently.


Hydrocortisone perturbs the cell proliferation pattern during feather morphogenesis: evidence for disturbance of cephalocaudal orientation. In this study, we have monitored the spatial distribution of S-phase cells during successive stages of normal feather morphogenesis using the specific marker BrdU. We also disturbed the development program by administration of hydrocortisone on the chorioallantoic membrane of 6.5-day chick embryos and examined the resulting pattern of BrdU incorporation. Our results show that a specific spatio-temporal pattern of cell proliferation occurs during successive stages of feather development and that this pattern accounts for the growth of feather buds according to the cephalocaudal orientation. Our experimental analysis showed that the stage-dependent alteration of feather morphogenesis (as shown by Züst, Ann. Embryol. Morphogen. 4, 1971 and confirmed by Démarchez et al., Dev. Biol. 106, 1984), is based on a stage-dependent alteration of the proliferation pattern in the epidermis. Forty-eight hours after treatment, non-induced epidermis ceases DNA synthesis and is unable to form placodes. Induced epidermis at the placodal and dermal condensation stages fails to produce the cohorts of S-phase cells responsible for the caudal outgrowth and the slanting shape of the buds. These young buds display anarchic proliferation in the whole epidermis possibly resulting in the appearance of "curly" feathers. Together, these results show the importance of the spatial pattern of ectodermal and mesodermal cell proliferation during the normal feather morphogenesis. Moreover, they corroborate the particular role of epidermis both in the establishment of feather rudiments and in the cephalocaudal orientation of the feathers.


Purification and localization of p10, a novel protein that increases in nymphal regenerating legs of Periplaneta americana (American cockroach). Content of a protein with a molecular mass of 10 kDa (p10) was found to increase significantly in regenerating legs of the American cockroach (Periplaneta americana). This protein was purified to homogeneity from a homogenate of regenerating legs. Partial amino acid sequencing indicated that p10 is a novel protein. Immunoblotting showed that its content in regenerating legs was 30 times that in normal legs, and decreased significantly when leg regeneration was complete. An immunofluorescence study revealed that p10 localizes exclusively on the external side of newly formed epidermis of regenerating legs.


Analysis of a unique molecule responsible for regeneration and stabilization of differentiated state of tissue cells. In Wolffian regeneration in the newt, a functional lens can be regenerated through cellular transdifferentiation of the pigmented epithelium of the mid-dorsal marginal iris. A novel monoclonal antibody, 2NI-36 mAb, generated in our laboratory has been utilized as a highly useful probe to study newt lens regeneration. The antigen molecule against this 2NI-36 mAb (2NI-36) became temporarily undetectable only at the site of lens regeneration. Moreover, the ventral iris pieces expressed the ability to differentiate a lens when pretreated with this monoclonal antibody and implanted in lentectomized eyes (Eguchi, Cell Differ. Dev. 25, Suppl., 1988). We have investigated the distribution of 2NI-36 in newt tissues. 2NI-36 was not specific to iris pigmented epithelium and distributed in many different kinds of mesodermal tissues, including dermis, blood vessel, mesonephros and so forth. 2NI-36 was also detected in either cell surface or intercellular spaces of cultured pigmented epithelial cells when they organized an epithelial cell sheet. Western blot analysis showed that 2NI-36 had the molecular weight of 50-200kD and was completely digested by trypsin, suggesting that 2NI-36 was a glycoprotein with many carbohydrate chains. It was also revealed by Western blot analysis that all the tissues in which 2NI-36 could be detected expressed this molecule similar to that in the iris epithelium. We expect that 2NI-36 is a glycoprotein expressed by various newt tissues and is functional to stabilize the differentiated state of each tissue cell in the same way as observed in the iris pigmented epithelial cells.


Expression and distribution of regeneration-responsive molecule during normal development of the newt, Cynops pyrrhogaster. We have investigated the expression and distribution of the regeneration-responsive molecule, 2NI-36, the loss of which is responsible for initiation of dedifferentiation of dorsal marginal iris pigmented epithelial cells to regenerate a lens. In the process of the normal development of the newt, the expression of 2NI-36 could not be detected in embryos at the early developmental stages, i.e., cleavages, gastrulation and neurulation, nor through later developmental stages to tail-bud, even though organogenesis was occurring. 2NI-36 was not detectable in any tissues until embryos reached developmental stage 40 (before hatching). In hatched larvae around developmental stage 46, strong expression of 2NI-36 was observed in several tissues including the vascular endothelium, the pigmented epithelium and the inner layer of skin epidermis. Moreover, 2NI-36 was present on the cell surface of these tissue cells. In conclusion, when the embryos hatch out to become swimming larvae that can feed by themselves, 2NI-36 begins to be expressed in some kinds of differentiated tissues. These results suggest that the function of 2NI-36 might be related to the completion of morphogenesis in development and also to the stabilization of the differentiated state of newly formed tissue cells.


Post-fertilization changes in Discoglossus pictus (Anura) eggs result in the formation of a capsular chamber where the egg rotates. Discoglossus pictus is one of the few anurans with an egg where a capsular chamber forms as a consequence of fertilization; the egg with its vitelline envelope rotates in this chamber according to gravity. We investigated the formation of the capsular chamber through various experimental cytochemical and ultrastructural approaches, and found that it is the product of plug liquefaction. The plug is a lens-shaped jelly coat typical of Discoglossus, and covering only part of the egg animal half. About 15 min after fertilization, granular material coming from the egg enters the plug, which gradually dissolves and, once liquefied, reorganizes itself around the entire egg, thus forming the chamber. This process goes through stages of rearrangement of the 25-A- and 250-A-thick filaments which constitute the plug matrix. The material entering the plug derives from the exocytosis of two vacuole types, with electron transparent and granular PAS-positive contents. Liquefaction of the plug correlates with the reduction of disulfide bonds present in its matrix. Furthermore, in vitro tests showed that the substances released from the egg are active in selectively dissolving only the plug, and lose activity upon boiling.


Alterations in biosynthetic accumulation of collagen types I and III during growth and morphogenesis of embryonic mouse salivary glands. We examined the biosynthetic patterns of interstitial collagens in mouse embryonic submandibular and sublingual glands cultured in vitro. Rudiments explanted on day 13 of gestation and cultured for 24, 48, and 72 h all synthesized collagen types I, III, and V. However, while the total incorporation of label into collagenous proteins did not change over the three-day culture period, the rate of accumulation of newly synthesized types I and III did change. At 24 h, the ratio of newly synthesized collagen types I:III was approximately 2, whereas at 72 h, the ratio was approximately 5. These data suggest that collagen types I and III may be important in initiation of branching in this organ, but that type I may become dominant in the later stages of development and in maintenance of the adult organ.


Relationship between innervation and forelimb regenerative capacity in the postmetamorphic pond frog Rana brevipoda porosa. The limb regenerative capacity and the quantity of innervation (the percentage of a cross-sectional area of amputation forelimb stump occupied by nerves) in the pond frog, Rana brevipoda porosa, was investigated in postmetamorphic froglets and adults of various sizes by means of amputating forelimbs through the zeugopodium. Nearly all the amputated limbs of newly metamorphosed froglets, 18-19 mm in snout-vent length, showed heteromorphic regeneration. However, the larger the body size, the lower the presence of limb regeneration. Limb regenerative capacity was completely lost in froglets and adults with snout-vents larger than 35 mm. The quantity of innervation of limbs was highest in newly metamorphosed froglets, gradually decreasing with growth. The nerve quantity in adults with a snout-vent length between 60-67 mm was approximately half that of the froglets. When the nerve supply was augmented by deviating ipsilateral sciatic nerve bundles to the forelimb stump, almost all limbs, which were usually non-regenerative with normal innervation, regenerated heteromorphically. These results show that the decline in limb regenerative capacity during postmetamorphic growth is in part attributable to the reduction in innervation levels to below the threshold level required for regeneration.


Study of yolk precursor transport in the avian ovary with the use of horseradish peroxidase. Ovaries of adult Japanese quails were exposed in vivo to the exogenous protein horseradish peroxidase (HRP) for varying lengths of time to investigate ultrastructurally the permeability of the wall of these follicles, the protein uptake capacity of granulosa and oocyte and the kinetics of protein uptake in different stages. There is a sudden increase in permeability of the follicle wall from previtellogenesis to vitellogenesis. This is not due to a loss of sealing (tight) junctions in the granulosa cell layer, but is probably related to a permeability change in the basement membrane. The transition from the slow growth phase to the rapid growth during vitellogenesis is accompanied by a limited widening of the intercellular channels and the concomitant development of a complex endocytotic apparatus in the ooplasm. The slowing down of yolk deposition during the last day before ovulation is accompanied by a narrowing of the intercellular channel width. The granulosa cells show a high intracellular HRP uptake during intermediary yolk formation. Transcytosis through the granulosa cannot be excluded but is probably a minor pathway at certain stages. The light microscopically detectable uptake of HRP by the oocyte coincides with the start of exogenous vitellogenesis. After 90 sec of exposure to HRP (intravenous injection) the tracer can be found in the intercellular channels of the granulosa and in superficially located yolk spheres. On the other hand it takes 10 min for the tracer to traverse the cortex of the oocyte.


N-glycosylated proteins interfere with the first cellular migrations in early chick embryo. Cell adhesion and migration properties which are known to play a crucial role in developmental events seem to be modulated by variations in glycosylation of glycoproteins. In the chick embryo, the extracellular matrix (ECM) appears as a loose meshwork of fibrillar material in the space between the epiblast and the hypoblast shortly before the first major cell migrations start. Chick embryos treated with tunicamycin (TN), a specific inhibitor of N-linked glycosylation of proteins, show little or no ECM, diminished cell adhesion and a dramatic alteration in the architecture of the epiblast and of the hypoblast. The first major cell migrations which signal the onset of PS and gastrula formation are inhibited irreversibly in these embryos. Tunicamycin induces a substantial change in the labeling pattern with change in mobility of some polypeptides and with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from these already present in the control blastoderm. The N-linked glycosylation of protein(s) that are synthesized during the interaction of the epiblast and of the hypoblast seem to play a critical role in cell adhesion and in the morphogenetic movements of gastrulation in the early chick embryo.


Unilateral renal agenesis in chick embryos: a model for chronic renal insufficiency. Although renal agenesis and dysgenesis are relatively common and significant birth defects, no animal model to date has been utilized to adequately study these developmental pathologies. Blockage of the migration of the mesonephric duct in Day 2 chick embryos results in unilateral renal agenesis (URA) on the operated side, thus providing a model of chronic renal insufficiency. Embryos with URA respond with an increase in the rate of growth of the remaining meso- and metanephric kidney. The allometric scaling of single (left) kidney weight to total body weight in control embryos is KM = 3.48M0.98 compared to KM = 3.02M1.16 in embryos with URA. In addition, embryos with URA exhibit a progressively polycystic mesonephros with distinct glomerulonephritis and expansion of the renal tubules. These renal changes are insufficient for normal urine (allantoic fluid) production and oliguria persists throughout incubation. While mortality is unaffected by URA in embryos up to Day 14 of incubation, there is a steady increase in mortality after Day 14; no chick embryo with URA lives beyond Day 18 of the 21-day incubation period.


The human placenta becomes haemochorial at the 13th week of pregnancy. Histological specimens of recent implantation sites are the basis of our current concept on human embryo implantation and placental development. In the Carnegie Collection maternal red blood cells were detected early in the primitive intervillous space (10th-12th day after conception). These cells were localized to the trophoblastic lacunae and originated from distended peripheral maternal sinusoids (Kaufmann, 1981). The classical theory states that progressively more and more maternal vessels are tapped. A true maternal blood flow is established around the 29th day. Dynamic investigations of human placental development in vivo are scarce and hampered by ethical considerations and the absolute requirement to refrain from using non aggressive and potentially harmful techniques. Despite these limitations such studies provide new insights that surprisingly contradict our previously and seemingly definitely established knowledge of the early phases of placental vascularization, and lead us to conclude that there is an absence of maternal blood circulation in the intervillous placental space (IVS) during the 12 first weeks of human pregnancy.


Advances in the detection of ploidy differences in cancer by in situ hybridization. Three main techniques allow the detection of changes in the cellular genomic content. The karyotyping procedure on metaphase spreads can give specific information on chromosome number and structural chromosome changes, but analyses are restricted to a limited number of chromosome spreads. Furthermore, cell culturing of (in particular solid) cancer specimens can result in selection of a minor tumour cell population with a high proliferative capacity. On the other hand, flow cytometry allows the analyses of large numbers of cells, but does not detect small variations in the DNA content or structural changes. The fluorescent in situ hybridization (FISH) procedure combines the advantages of the two former procedures, in that relatively large numbers of cells can be analysed easily and specific chromosomal changes can be detected.


Identification and fate of a marker chromosome in methotrexate-resistant V79,B7 cells by flow karyotyping and sorting, metaphase analysis and in situ hybridization. The chromosomes from a methotrexate (MTX)-resistant and its parental V79,B7 Chinese hamster cell line were analysed by the combined use of flow karyotyping and sorting, metaphase analysis and in situ hybridization with a probe for the dihydrofolate reductase (DHFR) gene responsible for methotrexate resistance. A marker chromosome with an elongated arm carrying the amplified DHFR gene was identified by in situ hybridization of metaphase cells of the methotrexate-resistant line. In the flow karyotype the marker chromosome was found as an additional peak with a higher DNA content compared with the largest chromosome of the sensitive line. This was additionally verified by G-banding of the chromosomes sorted from the marker peak. Several other chromosomal rearrangements not associated with the amplified gene could be identified in the methotrexate-resistant line by the combined use of flow karyotyping and metaphase analysis. The fate of the original marker chromosome was studied in cells growing several weeks in the absence of methotrexate, comparing flow karyotyping and metaphase analysis. The original marker chromosome was lost in about 50% of the cells after 5 weeks and in about 60% of the cells after 8 weeks; between 80 and 90% of the cells, however, contained marker chromosomes of various sizes. The MTX-resistance decreased in parallel during loss of the original marker chromosome. In conclusion, the study shows that the power of cytogenetic analysis is improved by the combined use of conventional cytogenetics, molecular cytogenetics and flow cytometry.


Spontaneous regression of residual tumour burden: prediction by Monte Carlo simulation. Current cancer treatment protocols are designed to release the tumour burden down to a small number of cells. In this study, we use Monte Carlo simulations to show that small populations of cells with intrinsic cell loss rates comparable to the cell loss rates observed clinically in human tumours, may regress spontaneously. Large populations of cells tend to grow under the same conditions of cell loss that result in extinction of small clones. Furthermore, minor variations in the intrinsic cell death probability near 0.50 result in large differences in the number of surviving cells calculated at the 100th generation. When Monte Carlo simulations of clonal growth resulted in clones with large populations (> 50 cells), the population as a whole behaved in a deterministic fashion (logarithmic growth) similar to those observed in clinically observed neoplasms and consistent with other published models of tumour growth. These findings provide a plausible explanation for the clinically observed failure of tumours to recur in instances where tumour burden remains following cancer therapy. The findings also demonstrate the usefulness of the Monte Carlo method to simulate biologic events in populations where the fate of each member of a population can be modeled probabilistically.


S phase, an evolutionary chromatin condensation state from G1 to G2, in a breast epithelial cell line. In order to better understand the changes in DNA organization during the cell cycle, we quantified the chromatin texture of breast epithelial cells and followed its evolution through a cell cycle. The diversity of quiescent cell states led us to limit this study to proliferating cell phases, and to choose a cell line with no G0 cells, the MDA AG cell line. We recently developed a methodology for characterizing in situ the cell cycle of breast epithelial cell lines using a cell image processor. This method is based on 15 densitometric and texture parameters computed on individual Feulgen-stained nuclei and on multiparametric analysis of the resulting data. Chromatin pattern assessment is based on nine texture parameters measured from grey-level co-occurrence and run-length section matrices. In the present study, texture parameter computation showed gradual and progressive modifications of nuclear texture. While discrimination of G1, G2 and M phases was possible, we could not discriminate G1 from S and S from G2. The chromatin pattern (defined by these nine parameters) in the G1 and early S phases, on the one hand, and in the late S and G2 phases, on the other hand, were similar. The parameter values of cells in the S phase progressively increased from G1 to G2. Two interphase chromatin condensation states were distinguished in these breast cells: a base state characteristic of a prereplicative stage and a very granular state characteristic of a postreplicative stage. We hypothesized that S cells are a blend of these two states, the evolution of a non-duplicated state toward a duplicated one.


Flow cytometric analysis of the cell cycle in chronic gastritis. Flow cytometric cell cycle analysis was recorded in gastric biopsy specimens from patients with normal gastric mucosa (GM), superficial gastritis (SG) and chronic atrophic gastritis (CAG). Cell-cycle analysis showed significantly higher percentages of cells in S- and S+G2/M-phase in CAG than in SG and normal GM (P < 0.0001). Moreover, CAG with severe or moderate atrophy showed significantly higher percentages of cells in S-phase (P < 0.05) and S+G2/M-phase (P < 0.02) than CAG with mild atrophy in antrum. In fundus, even if this increase was observed, it did not reach statistical significance. Consideration of concomitant pathologic findings such as oesophagite, gastric or duodenal ulcer, duodenite or benign polyp allowed a better differentiation of CAG both in antrum and in fundus. Significantly higher S-phase was observed in CAG with severe or moderate atrophy than in CAG with mild atrophy (P < 0.05). No statistically significant results were observed in patients with normal gastric mucosa or chronic gastritis and a concomitant pathologic finding.


Nuclear DNA ploidy in mammary carcinomas; using nuclear size as co-parameter reveals more complex patterns. Static DNA measurements using nuclear size as a co-parameter have been made on cell populations from 106 consecutive patients with proper imprints from fresh tumour tissues or fine-needle aspirates. When DNA content is plotted against the nuclear size of each individual cancer cell in a scatter diagram, different patterns are found. These patterns indicate the presence of subpopulations, which may be overlooked using conventional flow cytometric analysis or other techniques relying only on the nuclear DNA content. The presence of these subpopulations might, in addition, interfere with a correct definition and estimation of the percentage of S-phase cells.


Induction of AP-1 transcription factor components during T-cell activation by interleukin 1 and phorbol esters. We have examined the effect of interleukin 1 (IL-1) and phorbol esters [12-O-tetradecanoylphorbol-13-acetate (TPA)] on the expression of various components of the AP-1 transcription factor complex during T-cell activation. We previously found that a chloramphenicol acetyltransferase reporter gene driven by the collagenase TPA responsive element was expressed upon stimulation of T-cells by TPA and that this expression was enhanced when IL-1 was added as a costimulant; IL-1 alone had no effect on TPA responsive element-chloramphenicol acetyltransferase expression. In this study, we have found that stimulation of T-cells by IL-1 and TPA is accompanied by activation of a subset of immediate early genes that comprise the AP-1 transcription factor complex. junB and fosB were rapidly induced following stimulation with TPA. Although the levels of other fos-related mRNAs were also elevated, their maximal induction was delayed by approximately 5 h. IL-1 alone had little or no effect, but enhanced TPA induced transcription and steady-state levels of these mRNAs. The expression of fos and jun during T-cell activation was accompanied by increased specific binding of JunB, FosB, and fos-related antigen containing complexes to the TPA responsive element. These findings indicate that the synergistic effect of IL-1 and TPA on AP-1 mediated gene expression is due, in part, to the ability of IL-1 to enhance the expression of genes encoding specific AP-1 transcription factor components.(ABSTRACT TRUNCATED AT 250 WORDS)


Analysis of glutathione transferase P gene regulation with liver cells in primary culture. Glutathione transferase P (GST-P) gene is specifically and highly activated during rat chemical hepatocarcinogenesis. We have previously cloned the GST-P gene and have identified putative regulatory regions. To further explore regulatory mechanisms, deletion constructs of the GST-P gene fused to the chloramphenicol acetyltransferase (CAT) structural gene were introduced into primary cultured rat hepatocytes by electroporation, and their activity was determined. The expression of the GST-P-CAT fusion gene is quite low in these cells as compared to that in both a rat fibroblast cell line, 3Y1 cells, and a rat hepatoma cell line, dRLh84. The presence of the strong enhancer GPEI did not elicit any enhancing activity at its original position, or when it was located 3' of the CAT gene, although this element does enhance CAT activity significantly when located adjacent to the promoter. Cotransfection of neither c-jun nor c-fos expression vector, nor both vectors, could enhance the CAT activity, even though GPEI consists of two phorbol ester response element-like sites. Furthermore, the expression of jun family gene was not correlated with GST-P gene expression either in primary cultured hepatocytes or in hepatoma cell lines.


The ERGB/Fli-1 gene: isolation and characterization of a new member of the family of human ETS transcription factors. All cellular ets proteins contain a region of high amino acid identity to those found in the last two exons of the ets-1 gene (C domain). We have identified and characterized a new member of the human ETS gene family, ERGB. The ERGB gene shows extensive amino acid identity to the human ERG and the mouse Fli-1 genes. The ERGB gene is found to be transcriptionally active in a variety of human cell lines and tissues, in contrast to the more restrictive expression pattern of the ERG gene. The ERGB gene encodes for a 3.2-kilobase mRNA containing an open reading frame of 451 amino acids. The ERGB gene, like human ETS1, is located on chromosome 11 and is transposed to chromosome 4 as a result of the translocation t(4;11) associated with leukemia. Pulse-field gel analysis suggests that ETS1 and ERGB are more than 200 kilobases apart. Similar to the other members of the ets family (ets 1, ets 2), this new member is also able to trans-activate transcription of a reporter gene linked to the ETS-binding sequences derived from either the GATA-1 promoter or an optimal Ets-binding site.


Signal transduction defect appears to be the cause of rat prostate cancer cell fibroblast growth factor insensitivity. Using monolayer cultures of clonally isolated C3 and T5 rat prostate cancer cells, we determined that acidic (aFGF) and basic (bFGF) fibroblast growth factors profoundly enhanced T5 cell thymidine incorporation with half-maximum stimulation at 0.53 and 0.35 ng/ml, respectively. In contrast, aFGF or bFGF enhancement of C3 cell thymidine incorporation was about 5% of that of T5 cells, and effects were principally mitogen concentration independent. Saturation analyses and cross-linking studies established that both C3 and T5 cells contained high-affinity FGF receptors of 120 and 145 kilodaltons and that receptor content and Kd of C3 and T5 cells were comparable. aFGF or bFGF stimulation of T5 cell thymidine incorporation profoundly decreased as cell plating density was reduced from 1.5 x 10(5) to 1.0 x 10(4) cells/well. The modest response of C3 cells to either aFGF or bFGF also decreased as cell plating density was reduced. Because heparin preserves FGF biological activity and enhances bFGF binding to high-affinity FGF receptors, we examined the effect of heparin on FGF stimulation of C3 cell thymidine incorporation. We found that changes in cell plating density and/or medium heparin concentration had variable, inconsistent effects. These were C3 cell plating density associated and included inhibition or modest enhancement of FGF effects. Binding analyses established that high-affinity bFGF binding of C3 and T5 cells immediately prior to assessing FGF-stimulated thymidine incorporation was comparable and independent of cell plating density, implying that C3 cell FGF insensitivity was not attributable to differences in C3 and T5 cell FGF receptor content at the time of mitogen stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)


Myb expression is higher in malignant human colonic carcinoma and premalignant adenomatous polyps than in normal mucosa. Expression of the protooncogene c-Myb protein was assessed in normal mucosa and in tumor samples resected from six patients. We found that the tumor samples always expressed higher levels of full length Myb protein than the normal tissue. This contrasts with the situation in c-myb-associated hemopoietic malignancies of the mouse and chicken, in which Myb proteins are generally amino or carboxyl truncated. Tissues from five patients with colonic adenomatous polyps were also examined and found to express levels of Myb that were, in general, intermediate between those found in normal tissues and tumors. Of particular interest is that the more dysplastic polyps displayed higher Myb levels. In one patient with carcinoma and multiple colonic polyps, some polyps had intermediate levels of Myb, whereas one polyp with carcinoma in situ expressed tumor-like levels of Myb. To directly test the hypothesis that Myb expression may be important in determining the rate of colonic cell proliferation, we examined three colonic carcinoma cell lines and one polyp cell line. We found that the cell lines with the most rapid doubling times exhibited the highest Myb levels. In addition, we show that antisense myb oligonucleotides retard the proliferation of one of these colonic cell lines which expresses the highest level of Myb.


Protein kinase C beta gene expression is associated with susceptibility of human promyelocytic leukemia cells to phorbol ester-induced differentiation. To study the signal transduction pathway leading to phorbol 12-myristate 13-acetate (PMA)-induced differentiation in human promyelocytic HL-60 leukemia cells, we examined the expression of protein kinase C (PKC) isozyme genes in HL-60 cells that are susceptible or resistant to PMA-induced differentiation. The PKC-alpha, -beta, -gamma, -delta, epsilon, and -zeta transcript levels were assessed by Northern blotting, and the PKC-alpha, -beta, and -gamma protein levels were examined by immunoblotting. The PMA-resistant cell variants HL-525 and HL-534 were found to be deficient in the PKC-beta isozyme RNA and protein as compared with the PMA-susceptible HL-60 and HL-205 cell lines. In addition, a "delta-like" PKC RNA species identified in these cells demonstrated a reduced abundance in the HL-525 and HL-534 cells. Southern blot analysis indicated that the observed reduction in PKC-beta gene expression does not appear to be due to a gross deletion or rearrangement of the gene. The expression of the early response genes junB, c-fos, and c-jun was attenuated in PMA-treated HL-525 and HL-534 cells as compared to the PMA-treated HL-60 and HL-205 cells. These results suggest that the signal transduction pathway that leads to PMA-induced differentiation in the HL-60 cell system requires PKC-beta and/or delta-like PKC for the proper expression of the early response genes, and ultimately the expression of genes that define the mature state.


Growth factors, receptor kinases, and protein tyrosine phosphatases in normal and malignant melanocytes. Normal human melanocyte proliferation and differentiation is dependent on stimulation of one of three growth factor/receptor systems. They are fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and mast cell growth factor (MGF), which activate the FGF receptor, c-Met, and c-Kit, respectively, known to be receptor tyrosine kinases. In contrast, human melanoma cells from primary nodular and metastatic lesions grow autonomously partially because of inappropriate production of basic FGF (bFGF) and continuous activation of the bFGF-receptor kinase. Activation of transmembrane receptor tyrosine kinases in melanocytes stimulates not only proliferation but also the expression of pigmentation. Melanoma cells constitutively express several tyrosyl-phosphorylated proteins that in normal melanocytes are stimulated in response to growth factors. This high level of phosphorylation was not due to either the presence of constitutively active Kit kinase and Met kinase nor to the absence of any of several known protein tyrosine phosphatases. Because bFGF by itself does not transform melanocytes to melanomas, there must be additional cooperating factors that confer the malignant phenotype to pigment cells.


Proinflammatory and antiinflammatory cytokines in multiple sclerosis and central nervous system acquired immunodeficiency syndrome. While certain cytokines such as interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), IL-6, and transforming growth factor-beta (TGF-beta) may be involved in pro- and anti-inflammatory events in the central nervous system (CNS) of patients with multiple sclerosis (MS) and CNS acquired immunodeficiency syndrome (AIDS), there is not uniform consensus as to whether they are elevated or even detectable in all compartments of the body such as serum, cerebrospinal fluid (CSF), and tissue. Furthermore, if they are elevated in these diseases, there are no data as to whether they regulate the disease process itself. Myelin damage in MS is a punctate demyelination; in AIDS, it is a diffuse myelin pallor or dysmyelination. Oligodendrocytes are destroyed in MS but not CNS AIDS, suggesting a different mechanism for myelin loss in the two diseases. These different pathologies may provide clues about the role of macrophages, microglia, and/or the toxic products they produce in putatively giving rise to myelin damage. The stimuli that trigger such a destructive response by macrophages or glial cells and/or the regulation of the toxic events in the two diseases we would predict to be different. In MS, an effector cell-mediated lesion production and oligodendrocyte cell destruction seem to occur. We hypothesize that the effector is the inflammatory blood macrophage and/or microglial cell induced and promoted to its cytotoxic activity by a collaboration of neurotransmitters and cytokines. In CNS AIDS, virus-induced toxic products of glia and their diffusion through white and gray matter areas of the brain have been suggested. Such soluble mediators would then compromise metabolic processes of neurons and glia without widespread target cell loss.


Interactions of cytokines with the hypothalamus-pituitary axis. Major humoral mechanisms include the endocrine and immune systems, and there is substantial literature describing interactions between these systems during infection and inflammatory processes. Within the brain, such interactions are less well known. One major brain function altered during infection and inflammation and by several endocrine hormones is sleep. These changes in sleep provide a useful illustration of the interactions between cytokines and the hypothalamus-pituitary axis (HPA). Experimental evidence is reviewed that illustrates the interaction of cytokines, especially interleukin-1 (IL-1), with the HPA in regard to their effect on sleep. The evidence linking IL-1, growth hormone-releasing hormone/growth hormone, and corticotropin-releasing hormone to sleep regulation is reviewed. There is also evidence that shows that these two major sleep-regulatory systems are linked to each other.


Natural regulators of T-cell lymphokine production in vivo. The mammalian immune system possesses the intrinsic capacity to evoke a wide variety of functionally distinct effector mechanisms following stimulation by a particular antigenic substance. Such diversity in available responses is absolutely essential to the immunocompetent host, which must continually deal with a diverse set of potential pathogens within its ever-changing environment. The development of appropriate types of immune responses, therefore, represents a highly dynamic process that requires that an equivalent consideration be given to a large array of components, any one of which is capable of modulating the final outcome. While the nature and complexity of the antigen(s), plus the intracellular or extracellular mode of presentation, provide specificity and some selection to the developing process, the route of antigen entry, as well as the physiological status of the host at the time of antigen insult, also contribute significantly to the formation of any immune response. The overall objective of this article is to introduce the concept that platelet-derived growth factor (PDGF) (either preformed or synthesized in response to stimulation), plus a number of steroid hormones (some of which are end-organ metabolized at local tissue sites), can all play significant roles in the genesis of immunologic responses in vivo.


Presentation of three different viral peptides is determined by common structural features of the human lymphocyte antigen-A2.1 molecule. To assess whether similar or dissimilar molecular features of class I human lymphocyte antigen (HLA) molecules determine the presentation of structurally diverse peptides, we have examined the influence of different pockets within the HLA-A2.1 molecule on the presentation of three different viral peptides. The influenza virus M1 58-66, HTLV-I Tax peptide 12-19, and HCMV gB 619-628 are minimal peptides that induce HLA-A2.1-restricted non-cross-reactive CTL responses. The influence of distinct structural features of HLA-A2.1 on peptide presentation was analyzed using a panel of 14 HLA-A2 mutants each with single amino acid substitutions in one of six pockets that are located in the peptide binding site. Ten of the 14 mutants showed concordant effects on the presentation of all three peptides to their peptide-specific CTL lines. Four of the mutants had a negative effect on the presentation of only one or two of these viral peptides. These findings indicate that common structural features in HLA-A2 determine the binding and conformation of different peptides, and help to provide a plausible explanation for how diverse peptides bind to HLA-A2.


Immunobiology of primary murine melanomas. Primary cutaneous melanomas can be induced in inbred mice by applying a dose of dimethylbenz[a]anthracene to the skin of 4-day-old mice, and then applying repeated doses of a tumor promoter to the same site over a long period of time. Preliminary experiments suggest that the final incidence of melanomas is strongly influenced by the age at which the initiating dose of carcinogen is applied. Melanomas induced by this method in C3H mice are immunogenic and exhibit a high degree of cross-reactivity when tested by immunization and challenge in vivo. Exposing the mice to ultraviolet (UV) radiation during carcinogenesis dramatically accelerates the appearance of melanoma. We are attempting to determine how UV radiation potentiates melanoma induction by studying the growth of melanoma cells transplanted into UV-irradiated skin. Our studies suggest that UV irradiation accelerates the outgrowth of melanoma cells by means of a local, immunosuppressive effect on the skin. However, this effect is distinct from the ability of UV irradiation to alter epidermal Langerhans cells and interfere with the induction of contact hypersensitivity responses. We postulate that UV irradiation augments melanoma development by interfering with the efferent arm of the immune response in the UV-irradiated site.


Tumor-specific antigens of mouse tumors. This article reviews the serological analysis of cell surface antigens of chemically induced sarcomas of murine origin. This analysis, which was aimed at molecular characterization of the highly restricted tumor specific transplantation antigens of sarcomas, led to the initial biochemical identification and characterization of p53. The results of this early study clearly pointed to p53 as playing a key role in cellular proliferation and transformation. Current research of the tumor-specific or -associated antigens of chemically-induced sarcomas and melanomas of murine origin is also discussed.


The use of congenitally immunodeficient mice to study human tumor metastases and immunotherapy. Congenitally immunodeficient strains of mice have proven valuable in the development of relevant models to study human tumor biology, metastases, and immunotherapy. Local invasion and extensive multiorgan metastases in athymic mice have been obtained following orthotopic implantation or onplantation of histologically intact fragments of human tumors. In C.B-17 severe combined immunodeficient (SCID) mice or in triple immunodeficient, beige/nude/xid (BNX) mice, the development and spread of inoculated human leukemia/lymphoma and/or melanoma have mimicked, in some cases, those observed in patients. Reports of reconstitution of SCID and BNX mice with human myeloid or lymphoid cells have suggested that these models might be useful for the study of human immune responses to autologous tumors in vivo. The severe immunocompromised status of these mice have also led to evaluations of the therapeutic efficacy of adoptively transferred, tumor-reactive human T cells. In this report, we review the pertinent information currently available on the use of congenitally immunodeficient mice in studies of human cancer biology and treatment.


In vitro growth patterns of normal human melanocytes and melanocytes from different stages of melanoma progression. Based on the clinicopathological classification of distinct stages of tumor progression in the melanocytic system, we have investigated the in vitro growth patterns and requirements of normal melanocytes and melanocytes isolated from different lesions of melanoma progression. Normal melanocytes depend on a combination of insulin-like growth factor (IGF-I) or insulin, 12-O-tetradecanoyl phorbol-13-acetate (TPA), alpha-melanocyte stimulating hormone (alpha-MSH), and basic fibroblast growth factor (bFGF) for in vitro proliferation. Nevus cells display a reduced need for TPA and are largely independent of bFGF. Both melanocytes and nevus cells have a finite lifespan in vitro and show no spontaneous transformation, whereas melanoma cells can be grown indefinitely in vitro. Cells from primary melanomas require only IGF-I or insulin for continuous growth, and metastatic melanoma cells can proliferate in base medium without addition of any growth factors or proteins. This progressive growth autonomy is paralleled by an increased competence for endogenous growth factor production. Among these growth factors, bFGF and melanoma growth-stimulatory activity (MGSA) act in an autocrine fashion. Melanoma-derived growth factors without apparent autocrine function, such as platelet-derived growth factor A and B (PDGF-A and PDGF-B) and transforming growth factor-alpha (TGF-alpha), might still be important for melanoma growth by stimulating surrounding normal fibroblasts, endothelial cells, or keratinocytes to secrete growth-promoting factors. The significance of growth factors such as transforming growth factor-beta (TGF-beta) and melanoma-inhibiting activity II (MIA II), which have a potentially negative autocrine function, remains unknown. The successful propagation of melanocytic cells of all stages of melanoma progression has yielded valuable insight into the mechanisms of growth regulation and malignant transformation.


Recognition of shared melanoma antigens by human tumor-infiltrating lymphocytes. We have established that melanomas express shared tumor antigens (Ags) that can be recognized by T cells if presented in the context of self-MHC molecules. Tumor-infiltrating lymphocytes (TILs) from six melanoma patients were tested for lysis of large panels of HLA-matched or unmatched targets representing a variety of tissue types. Lysis was specific for allogeneic melanomas sharing at least one HLA-A, -B, or -C Ag with TILs, and demonstrated commonly expressed tumor Ags. Similar findings were obtained when cytokine secretion by TILs was used to indicate specific Ag recognition. Transfection of the HLA-A2.1 gene into HLA-A2- melanoma lines conferred susceptibility to lysis by HLA-A2 restricted melanoma TILs, demonstrating expression of common tumor Ags among patients of diverse HLA types. These findings have important implications for developing broadly applicable cancer immunotherapies such as vaccines.


The effect of environmental temperature and humidity on 24 h energy expenditure in men. The effects of environmental temperature and humidity and their interaction on 24 h energy expenditure were measured using whole-body indirect calorimetry in eight normal-weight young men who wore standardized light clothing and followed a controlled activity regimen. A randomized-block experimental design was used, with temperature effects assessed by measurements at 20, 23, 26 and 30 degrees, while humidity was altered from ambient (50-65% relative humidity) to high (80-93% relative humidity) at 20 and 30 degrees only. There was no significant effect of humidity on 24 h energy expenditure at the two extreme temperatures in this range, though when periods of sleep and exercise were excluded the energy expenditure at high humidity was significantly higher than that ambient humidity (P < 0.02). The effect of temperature at ambient humidity levels showed lower values at 23 and 26 degrees than at 20 and 30 degrees (P < 0.02). The effect of temperature was not equally apparent in all components of the 24 h energy expenditure, as sleeping metabolic rate and the energy cost of walking and cycling showed no significant effect of temperature over this range. This raises the possibility that the effects of temperature are attributable to behavioural changes during the waking portion of the day rather than any non-shivering thermogenic mechanisms at tissue level.


Compensatory growth in broiler chicks fed on Lemna gibba. The growth of broiler chickens on diets containing various levels of Lemna gibba was evaluated. Groups of broiler chicks were fed on diets containing 0-400 g Lemna gibba/kg for 3 weeks. These chickens were then changed to standard diets for a further 2 weeks. As the level of Lemna gibba increased, feed consumption and weight gain decreased. However, when diets were changed to the standard diet, compensatory growth was observed. In a second experiment, diets were formulated with a metabolizable energy of 5.02 MJ (1200 kcal)/kg Lemna gibba and included a finer-milled Lemna gibba. Chickens were fed on diets containing 0-300 g Lemna gibba/kg for 4 weeks. Each group was then divided into two subgroups. For the next 2 weeks one of these sub-groups was maintained on the experimental (Lemna gibba) diets (LL), while the other sub-group was changed to a standard diet (LS). Bird fed at levels above 150 g Lemna gibba/kg had decreased consumption and weight gain. These birds when changed to a standard diet tended to have increased weight gain compared with chickens continuously fed standard rations. LS birds had significantly higher weight gains and feed consumption and lower feed conversion than LL birds. In contrast to older birds, chicks fed on Lemna gibba at high concentrations showed growth retardation. When changed back to a standard diet they demonstrated normal or compensatory growth.


Effects of repeated gestation and lactation on milk n-6 fatty acid composition in rats fed on a diet rich in 18:2n-6 or 18:3n-6. The present study examined the effect of repeated gestation and lactation on the levels of long-chain n-6 polyunsaturated fatty acids in rat milk fat, and examined whether such levels might be modulated by supplementing the diet of the lactating dams with either (g/kg) 50 safflower oil (SFO; containing 800 g 18:2n-6/kg), or 50 evening primrose oil (EPO; containing 720 g 18:2n-6 and 90 g 18:3n-6/kg). The milk was collected at three different times (days 1, 8 and 15) in each given lactation period from female Sprague-Dawley rats which were successively bred for four pregnancies and lactations. Results showed that dietary fat and breeding frequency had no significant effects on milk triacylglycerol content, but they modified the pattern of milk fatty acids in both triacylglycerol and phospholipid fractions. After three or four successive breedings rats fed on EPO produced milk containing less saturated but more monounsaturated and polyunsaturated fatty acids compared with those fed on SFO. During the course of lactation the levels of n-6 metabolites, e.g. 18:3n-6, 20:3n-6 and 20:4n-6, in milk fat declined progressively. However, they were consistently higher in the EPO group than in the SFO group. These findings suggest that the levels of long-chain n-6 metabolites in the milk fat may be increased through supplementing the maternal diet with 18:3n-6.


The intake, digestion and protein degradation of grazed herbage by early-weaned lambs. Sixty-four intact lambs and twenty-four lambs fitted with a duodenal cannula were weaned at 6 weeks of age and grazed pure species swards of either lucerne (Medicago sativa), white clover ((Trifolium repens), ryegrass (Lolium perenne) or prairie grass (Bromus catharticus) for 6 weeks. Intake and duodenal digesta flow were estimated when lambs were 8 and 12 weeks of age. Lambs grazing the two legume species grew at a similar rate, as did lambs grazing the two grass species. Legumes promoted a 38% higher growth rate than grasses. The higher growth rate of lambs grazing legumes was associated with a 36% higher digestible organic matter intake (DOMI; 29.5 and 21.7 g/kg body-weight per d for legume and grass respectively) and a 33% higher duodenal non-ammonia-nitrogen (NAN) flow (1.22 and 0.92 g/kg body-weight per d respectively). There was no species difference in the site of organic matter digestion; on average 0.56 of DOMI was apparently digested in the rumen and 0.77 of DOMI was truly digested in the rumen. There was no difference in duodenal NAN flow, relative to DOMI (average, 43 g/kg) or to organic matter apparently digested in the rumen (80 g/kg). Similarly, there was no difference in microbial N flow relative to duodenal NAN (0.50 g/g) and organic matter apparently (41 g/kg) or truly (29 g/kg) digested in the rumen. It was concluded that the higher growth rates achieved by lambs grazing legumes were due to higher intakes which increased the total quantity of nutrients supplied despite more protein being lost in the rumen of lambs consuming legumes.


Acetylation of peptides inhibits their degradation by rumen micro-organisms. Proteins and peptides were acetylated using acetic anhydride in order to block their N-terminal amino groups and thereby to prevent their hydrolysis by rumen microbial aminopeptidases. The effects of acetylation on peptide breakdown and ammonia production were determined by incubating unmodified and acetylated substrates with sheep rumen micro-organisms in vitro. Ammonia production from casein and lactalbumin was affected little by acetylation, but acetylation of the corresponding enzymic hydrolysates caused ammonia production to be more than halved after 3-6 h incubation. Estimation of peptides remaining in rumen fluid showed that the decreased ammonia production was a consequence of peptides being hydrolysed more slowly. Acetylated Ala-Ala, Ala-Ala-Ala (Ala3), Leu-Gly-Gly, Phe-Gly-Gly and Val-Gly-Ser-Glu survived incubation with rumen fluid in vitro for 6 h, whereas almost none of the corresponding unmodified peptides was present at 6 h. The protection afforded to larger pure peptides was less reliable: for example, 72% of acetylated bradykinin was hydrolysed after 1 h. N-Acetyl Ala3 had only a minor inhibitory effect on the breakdown of Ala3 and Ala4, suggesting that although acetyl peptides were broken down more slowly than unmodified peptides they did not inhibit peptidase activity.


Responses in tissue protein synthesis to sub- and supra-maintenance intake in young growing sheep: comparison of large-dose and continuous-infusion techniques. In ten lambs (average live weight 33 kg), five offered 300 g/d (approximately 0.6 x maintenance; L) and five 900 g/d (1.8 x maintenance; H), tissue protein synthesis was measured by three procedures simultaneously. The techniques involved continuous infusion of [U-14C]phenylalanine and [1-13C]leucine over 7-8 h followed by a terminal large dose of [15N]phenylalanine during the last 30 or 60 min. Rates of protein synthesis were then calculated based on the free amino acid or oxo-acid isotopic activity in either arterial, iliac venous blood or tissue homogenate for the continuous-infusion studies, or on plasma or tissue homogenate for the large-dose procedure. For muscle (> 99%), and to a lesser extent skin (85-93%), effective flood conditions were achieved with the [15N]phenylalanine but were either not established or maintained for liver and tissues of the gastrointestinal tract (< 50%). The large dose of phenylalanine also caused changes in the concentration and isotopic activity of blood leucine and 4-methyl-2-oxo-pentanoate. Based on the assumption that the large-dose procedure yields the closest value for the true rate of protein synthesis (L 1.97%/d, H 2.85%/d) then, for muscle, only values based on the homogenate as precursor gave comparable results for both leucine (L 1.83%/d, H 3.01%/d) and phenylalanine (L 1.67%/d, H 2.71%/d) continuous infusion. The values based on the arterial or venous amino or oxo-acid were significantly less, more so at the lower intake. In contrast, for skin, a tissue dominated by export protein synthesis, values from the large-dose procedure (L 6.37%/d, H 10.98%/d) were similar to those derived with arterial or venous metabolites as precursor (L 5.23 and 6.93%/d, H 9.98 and 11.71%/d for leucine), but much less than those based on homogenate data. Based on the large-dose technique, protein synthesis increased with intake in muscle (P < 0.001), skin (P = 0.009) and liver (26.7 v. 30.5%/d; P = 0.029). The contributions of muscle and skin to total protein synthesis were approximately equal. The incremental efficiency of conversion for muscle of synthesized protein into deposition appeared to be similar to values reported for rodents.


Effect of food intake on hind-limb and whole-body protein metabolism in young growing sheep: chronic studies based on arterio-venous techniques. Whole-body protein synthesis, estimated by the irreversible loss rate procedure, and hind-leg protein metabolism determined by arterio-venous techniques were monitored in response to three nutritional conditions (approximately 0.6, 1.2 and 1.8 x energy maintenance (M)) in ten wether lambs (33 kg average live weight). In all lambs and treatments measurements were based on radiolabelled phenylalanine, but the terminal procedures (five at 0.6 x M and five at 1.8 x M) also included infusion of [1-13C]leucine; this permitted comparison of amino acids catabolized (leucine) and non-metabolized (phenylalanine) by the hind-limb tissues. Whole-body protein synthesis increased with intake and the relationship with energy expenditure was slightly lower than that reported previously for pigs and cattle. The efficiency of protein retention:protein synthesis did not exceed 0.25 between the two intake extremes. Effects of intake on amino acid oxidation were similar to those observed for cattle. Hind-limb protein synthesis also increased significantly (P < 0.001) in response to intake. Estimates of protein gain, from net uptake values, indicated that the tissues made a greater proportional contribution to total protein retention above M and to protein loss below M, emphasizing the role played by muscle tissue in providing mobile protein stores. The rates of protein synthesis calculated depended on the selection of precursor (blood) metabolite, but rates based on leucine always exceeded those based on phenylalanine when precursor from the same pool was selected. The incremental efficiency of protein retained:protein synthesis was apparently unity between 0.6 and 1.2 x M but 0.3 from 1.2 to 1.8 x M. Blood flow through the iliac artery was also proportional to intake. Leucine and oxo-acid catabolism to carbon dioxide increased with intake such that the metabolic fate of the amino acid was distributed in the proportion 2:1 between protein gain and oxidation. The rates of oxidation were only 1-3% the reported capacity of the rate-limiting dehydrogenase enzyme in muscle, but sufficient enzyme activity resides in the hind-limb adipose tissue to account for such catabolism.


Impairment of cysteine synthesis from methionine in rats exposed to surgical stress. The activity of liver cystathionase (EC 4.4.1.1) was decreased after 3 d of stress induced by surgery. The rate of L-cysteine synthesis from L-methionine was significantly higher in isolated hepatocytes from controls than in hepatocytes from rats suffering from surgical stress. The half-life of L-[2(n)-3H]methionine was significantly higher in rats submitted to surgical stress than in controls. Plasma L-methionine:L-cystine ratio was higher in stressed rats than in controls. L-cystine uptake was significantly increased in the surgically-stressed rats when compared with the controls. All these facts are consistent with the hypothesis that the observed inhibition of cystathionase is physiologically important and that L-cysteine might be considered as an essential amino acid in cases of surgical stress.


The cholesterol-raising effect of coffee in the Syrian hamster. Adult male Syrian hamsters were fed on a high-fat diet with or without access to boiled coffee. Plasma total, low-density-lipoprotein- and high-density-lipoprotein-cholesterol and triacylglycerol concentrations were increased by the coffee and very-low-density-lipoprotein-cholesterol concentrations were lowered. It is concluded that the Syrian hamster is a suitable animal model in which to study the hypercholesterolaemic effect of coffee.


Lipid composition and fluidity of the erythrocyte membrane in copper-deficient rats. The influence of dietary copper on lipid composition, phospholipid fatty acid and protein profiles and fluidity of the erythrocyte membranes of rats is reported. In general Cu deficiency in rats induced some changes in the phospholipid-fatty acid profile of erythrocyte membranes when compared with Cu-adequate animals. Stearic (18:0) and docosadienoic (22:2n-3) acids contents, for example, were significantly increased (P < 0.001) while oleic (18:1n-9) and linolenic (18:3n-3) acid contents were significantly depressed (P < 0.001) as a result of Cu deficiency. Moreover the cholesterol:phospholipids molar ratio and the cholesterol (mol):membrane proteins (mg) ratio in Cu-deficient rats were, to different degrees, significantly lower than in animals fed on Cu-adequate diets. In addition, diets deficient in Cu led to a reduction in erythrocyte membrane fluidity (P < 0.001) as assessed by the intramolecular excimer fluorescence of 1,3-di(1-pyrenyl) propane. However, no significant alteration in the phospholipid:protein ratio was observed as a result of differences in dietary treatment. The pattern of erythrocyte membrane proteins obtained with sodium dodecyl sulphate-polyacrylamide gel electrophoresis did not seem to be influenced by Cu-deficient diets.


Glycaemic index of conventional carbohydrate meals. The glycaemic index (GI) and the triacylglycerol response were measured in thirty non-insulin-dependent diabetes mellitus patients given 50 g portions of five different conventional Indian meals containing semolina (Triticum aestivum) cooked by two different methods, or combinations of semolina and pulse (black gram dhal (Phaseolus mungo), green gram dhal (Phaseolus aureus) or Bengal gram dhal (Cicer arietum)). There were no significant differences among meals in mean GI except for meals based on roasted semolina or semolina-black gram dhal. Compared with the blood glucose response for a 50 g glucose load, only meals based on steam-cooked semolina and semolina-Bengal gram dhal elicited a significantly lower response at 1 h postprandially, and only meals based on semolina-black gram dhal at 2 h postprandially. No significant differences were found among the meals in the triacylglycerol-response.


The effects of feed intake and purified cellulose on the endogenous ileal amino acid flow in growing pigs. The effects of level of feed intake (0.8, 1.2 and 1.6 kg/d) and body-weight of the pig (49 and 92 kg) in Expt 1, and dietary neutral-detergent fibre (NDF; 30, 60, 90, 120 and 150 g/kg) in Expt 2 on the endogenous ileal flow of amino acids (AA) and nitrogen were studied with protein-free diets into which purified wood cellulose was incorporated at the expense of maize starch. In Expt 1, one of the protein-free diets containing 90 g NDF/kg was used. Female pigs were fitted with a simple 'T' cannula at the terminal ileum. In Expt 1, the endogenous ileal AA and N flow, expressed as g/kg dry matter (DM) intake, decreased significantly (P < 0.05) with increasing DM intake, except for proline. By contrast, the values expressed as g/d remained constant. There was no significant difference in endogenous ileal flow (P > 0.05) between initial body-weights of 45 and 90 kg except for histidine, isoleucine, phenylalanine, threonine, valine and serine, in which the ileal flow determined at the higher body-weight was significantly higher (P < 0.05). In Expt 2, the effects of dietary cellulose levels on the endogenous ileal flow of AA and N were not significant (P > 0.05), although the values tended to increase as dietary cellulose levels increased for most AA and for N. It is concluded that the daily endogenous ileal flow of AA and N remains relatively similar at different DM intake and cellulose levels. Therefore, correction of apparent ileal digestibility of AA and N to the true ileal digestibility should be made with the endogenous ileal flow values expressed on a daily amount basis, not the values expressed on a DM intake basis.


The effects of ispaghula on rat caecal fermentation and stool output. The colonic fermentation of ispaghula, a mucilage from Plantago ovata composed mainly of arabinoxylans, and its effects on stool output and caecal metabolism were investigated. Four groups of eight rats were fed on a basal diet (45 g non-starch polysaccharides/kg) for 28 d. The diet was then supplemented with ispaghula (g/kg; 0, 5, 15 or 50) for 28 d. Ispaghula increased stool dry weight and apparent wet weight but faecal water-holding capacity (amount of water held per g dry faecal material at 0.2 mPa) was unchanged. The extent of faecal drying in the metabolism cages was measured for rats fed on the basal diet and 50 g ispaghula/kg diet. At the faecal output levels encountered, only an 8% loss of wet weight would be predicted over 24 h and this was independent of diet. Faecal short-chain fatty acid (SCFA) concentration did not change but SCFA output increased. The molar proportion of SCFA as propionic acid increased and faecal pH was reduced. Values from pooled faecal samples suggested that approximately 50% of the ingested ispaghula was excreted by the 50 g ispaghula/kg diet group. Diaminopimelic acid (a constituent of bacterial cells) concentrations fell but output was unchanged indicating no change in bacterial mass. Similar changes were seen in the caecal contents but caecal pH and SCFA were unaffected. Ispaghula increased both caecal and colonic tissue wet weight and colonic length. Our results suggest that ispaghula is partly fermented in the rat caecum and colon, and loses its water-holding capacity. However, it is still an effective stool bulker and acts mainly by increasing faecal water by some unknown mechanism.


Comparison of the effects of cassava (Manihot esculenta Crantz) organic cyanide and inorganic cyanide on muscle and bone development in a Nigerian breed of dog. Effects of cassava (Manihot esculenta Crantz)-borne organic cyanide and inorganic cyanide in the form of sodium cyanide on bone and muscle development were investigated in eighteen dogs of Nigerian breed. After 16 weeks of stabilization in the laboratory from the time of purchase when the dogs were fed on the same diet, they were randomly assigned to three experimental groups of six dogs each. The control group was fed on rice while the other two groups were fed on either cassava (gari) or rice plus cyanide. The three diets were made isoenergetic and isonitrogenous by varying the quantity of meat incorporated into them. The results obtained after 14 weeks of feeding the respective diets indicated that there was retardation of muscle development in the gari-fed dogs. This may have resulted from gluconeogenesis from muscle protein associated with suppression of production of insulin by the pancreas in this group. The results indicated also that the effects of inorganic dietary cyanides on muscle development were different. Both forms of dietary cyanides, however, had no adverse effect on bone development.


Nutritional outcome and immunocompetence in mice fed on a diet containing raw field beans (Vicia faba, var. minor) as the source of protein. Feeding growth mice on diets containing raw field beans (Vicia faba var. minor) as the only source of protein brought about an impairment in growth, muscle mass and liver weight. No changes in food consumption were observed, but the food intake:weight gain ratio was increased in those animals. Plasma protein, triacylglycerols and cholesterol values were not affected by the dietary treatment although serum glucose and zinc levels fell after legume intake as well as the number of circulating erythrocytes. The relative enlargement of thymus and spleen in the legume-fed mice was apparently accompanied by a reduction in the cell number and an increase in cell size, while the protein synthesis capacity followed differentiated patterns in both tissues when assessed through protein, DNA and RNA determinations. The haemagglutination titres and the number of rosette-forming cells were lower in those animals fed on the field bean diet as well as the splenic lymphocyte responses to phytohaemagglutinin, Concanavalin A or lipopolysaccharide mitogens used to evaluate the functional status of T and B lymphocytes. The present study describes, apparently for the first time in mice, the involvement of field bean intake in some immunological disturbances affecting both humoral- and cell-mediated aspects of the immune response.


Influence of low dietary lipid content on anorexia and [14C]glucose uptake in the intestine of zinc-deficient mice. Zinc deficiency was induced in adult male mice by feeding them for 8 weeks on a purified semi-synthetic Zn-deficient diet (ZD) containing 90 g lipid/kg (60 g maize oil plus 30 g cod-liver oil). One group was then fed on a low-lipid Zn-deficient diet (ZDLR) containing 30 g cod-liver oil/kg as the sole lipid source for a further 8 weeks. At the end of the experiment the stomach clearance rate, daily food intake, body-weight gain and [14C]glucose uptake in the intestine were significantly higher in group ZDLR than in mice that continued eating the Zn-deficient lipid-adequate diet ZD, and were comparable to results for a group given a Zn-supplemented diet. These results suggest that the pathogenesis of anorexia, nutrient malabsorption and growth retardation are secondary to lipid malabsorption resulting from Zn deficiency.


Erythrocytes, erythrocyte membranes, neutrophils and platelets as biopsy materials for the assessment of zinc status in humans. During a controlled zinc depletion-repletion study, fifteen men aged 25.3 (SD 3.3) years were fed on a low-Zn diet with high phytate:Zn and phytate x calcium:Zn molar ratios for 7 weeks, followed by a 2 week repletion period when 30 mg supplemental Zn/d was given. Changes in plasma, urine, and hair Zn concentrations, taste acuity, and cellular immune response confirmed the development of mild Zn deficiency. Zn concentrations in neutrophils, platelets, erythrocytes and erythrocyte membranes, mean platelet volume, and activities of alkaline phosphatase (EC 3.1.3.1) and alpha-D-mannosidase (EC 3.2.1.24) in neutrophils did not respond to changes in Zn status. In contrast, alkaline phosphatase activity in erythrocyte membranes showed a significant decline which was consistent in all subjects (nmol product formed/min per mg protein; baseline v. 7-week Zn depletion, 0.656 (SD 0.279) v. 0.506 (SD 0.230), at 7 weeks; P < 0.05); neutral phosphatase activity remained unchanged. Alkaline phosphatase activity in erythrocyte membranes may be a potential index of Zn status in humans.


Efficacy of massive oral doses of retinyl palmitate and mango (Mangifera indica L.) consumption to correct an existing vitamin A deficiency in Senegalese children. Administration of large oral doses of retinyl palmitate has become the most widely practised vitamin A deficiency prevention strategy in developing countries. We conducted a follow-up study among 220 Senegalese children aged 2-7 years suffering from moderate undernutrition to determine the efficacy of vitamin A treatment on their vitamin A status assessed by biochemical and cytological (impression cytology with transfer) methods. The first examination (T = 0 m[onth]) was carried out during April 1989, before the mango (Mangifera indica L,) harvest. The second examination (T = 2 m) was carried out 2 months after vitamin A treatment during June 1989 when ripe mangoes become widely available. Conjunctival cells of the eyes of the children with or without ocular inflammation were responsive to vitamin A administration (P < 0.01). There was a significant increase (P < 0.001) in mean serum retinol and beta-carotene levels between T = 0 m and T = 2 m. Mean serum retinol-binding protein (RBP) and transthyretin (TTR) levels did not differ significantly (P > 0.05) at T = 0 m and T = 2 m. Despite the intake of vitamin A, 54% of the children who had abnormal cytology at T = 0 m remained abnormal at T = 2 m. This was due to inadequate levels of TTR and RBP, presumably due to the cereal diet eaten by the Senegalese population. children with abnormal eye cytology had lower serum retinol levels than those with normal eyes at T = 0 m, and beta-carotene values did not correlate with eye cytological abnormalities at T = 0 m. Children with normal cytology had higher serum retinol and also beta-carotene levels than those with abnormal cytology after massive oral doses of vitamin A and consumption of mangoes at T = 2 m. Retinyl palmitate may, therefore, only lead to partial cytological improvement due to a lack of retinol-carrier proteins but dietary beta-carotene may also be involved.


Importance of specific adenosine N7-nitrogens for efficient cleavage by a hammerhead ribozyme. A model for magnesium binding. Five modified hammerhead ribozyme/substrate complexes have been prepared in which individual adenosine N7-nitrogens have been excised. The modified complexes were chemically synthesized with the substitution of a single 7-deazaadenosine (c7A) base analogue for residues A11, A14, A26, A27, or A28. Two of the base analogues, c7A11 and c7A14, occur in a 19-mer ribozyme, while the remaining three residues, c7A26, c7A27, and c7A28, are present in a 24-mer substrate. Under stoichiometric conditions, four of the complexes are cleaved with relatively little change in rate when compared with that of the native complex. However, the relative rate for the c7A11 complex is some 35-fold slower than that of the native complex. Steady-state kinetic analyses indicate that the cleavage efficiencies, as measured by kcat/KM, for the c7A14, c7A26, c7A27, and c7A28 complexes are reduced 18-fold, 10-fold, 34-fold, and 16-fold, respectively. These reductions in cleavage efficiency are primarily a result of lower kcat values. By comparison, the cleavage efficiency of the c7A11 complex is reduced more than 200-fold relative to that of the native complex, again primarily as a result of a lower kcat value. The results suggest that the N7-nitrogen of A11 in the hammerhead ribozyme/substrate complex is critical for efficient cleavage activity. The results of the present work, in combination with those from previous reports, indicate that five critical functional groups are located within the tetrameric sequence G10A11U12G13. A preliminary model for the binding of a single magnesium cofactor to this portion of the sequence is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)