Circadian and Ultradian Rhythms in Blood Glucose and Plasma Insulin of Healthy Adults

The circadian and ultradian variations of blood glucose and plasma insulin have been characterized individually and as a group phenomenon in five healthy young adults studied while adhering as closely as possible to their usual routine of sleep, activity, meal content and timing. Three complementary methods were used to analyze the data: displaying raw data as a function of time; cosinor method according to Nelson and Halberg; and time series analyses as proposed by De Prins and Malbecq. The subjects were studied in the laboratory and their life routine were controlled, but very close to that of their habitual routine. They had mainly ultradian rhythms of blood glucose (mainly about 6 hr) and circadian rhythms of immunoreactive insulin (I.R.I.). Blood glucose ultradian rhythms seem to be mainly but not exclusively mealtime dependent, while I.R.I, circadian rhythms appear to be primarily endogenous in origin. Therefore, the role played by insulin in the control of blood glucose levels seems to be programmed on a circadian basis rather than by a time independent feedback phenomenon as postulated by the conventional homeostatic hypothesis. The advantage of this chronophysiologic approach is to consider circadian rhythms of both I.R.I. and insulin effectiveness as an adaptive phenomenon able to maintain blood sugar changes in the ultradian domain of rhythms.     

Comparative analysis of the intracellular localization of c-Myc, c-Fos, and replicative proteins during cell cycle progression.

In eukaryotic cells, nucleus-cytoplasm exchanges play an important role in genomic regulation. We have analyzed the localization of four nuclear antigens in different growth conditions: two replicative proteins, DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), and two oncogenic regulatory proteins, c-Myc and c-Fos. A kinetic study of subcellular localization of these proteins has been done. In cultures in which cells were sparse, these proteins were detected in the nucleus. When proliferation was stopped by the high density of culture cells or by serum starvation, these proteins left the nucleus for the cytoplasm with different kinetics. DNA polymerase alpha is the first protein to leave the nucleus, with the PCNA protein, c-Fos, and c-Myc leaving the nucleus later. In contrast, during serum stimulation c-Fos and c-Myc relocalize into the nucleus before the replicative proteins. We also noticed that in sparse cell cultures, 10% of the cells exhibit a perinuclear staining for the DNA polymerase alpha, PCNA, and c-Myc proteins but not for c-Fos. This peculiar staining was also observed as an initial step to nuclear localization after serum stimulation and in vivo in Xenopus embryos when the G1 phase is reintroduced in the embryonic cell cycle at the mid-blastula stage. We suggest that such staining could reflect specific structures involved in the initiation of the S phase.     

Nested chromosomal fragmentation in yeast using the meganuclease I-Sce I: a new method for physical mapping of eukaryotic genomes.

We have developed a new method for the physical mapping of genomes and the rapid sorting of genomic libraries which is based on chromosome fragmentation by the meganuclease I-Sce I, the first available member of a new class of endonucleases with very long recognition sequences. I-Sce I allows complete cleavage at a single artificially inserted site in an entire genome. Sites can be inserted by homologous recombination using specific cassettes containing selectable markers or, at random, using transposons. This method has been applied to the physical mapping of chromosome XI (620 kb) of Saccharomyces cerevisi and to the sorting of a cosmid library. Our strategy has potential applications to various genome mapping projects. A set of transgenic yeast strains carrying the I-Sce I sites at various locations along a chromosome defines physical intervals against which new genes, DNA fragments or clones can be mapped directly by simple hybridizations.     

Competitive exclusion of diarrheagenic Escherichia coli (ETEC) from human enterocyte-like Caco-2 cells by heat-killed Lactobacillus

Diarrheagenic Escherichia coli (ETEC) bearing CFA/I or CFA/II adhesive factors specifically adhere onto the brush border of the polarized epithelial human intestinal Caco-2 cells in culture. Heat-killed Lactobacillus acidophilus strain LB, that adheres onto Caco-2 cells, inhibits diarrheagenic Escherichia coli adhesion in a concentration-dependent manner. Since the L. acidophilus does not express ETEC-CFA adhesive factors, it can be postulated that the heat-killed L. acidophilus LB cells inhibit diarrheagenic E. coli attachment by steric hindrance of the human enterocytic ETEC receptors.     

Enhancement of shoot regeneration potential by liquid medium culture from mature cotyledons of sunflower (Helianthus annuus L.)

We describe here a liquid culture system for the regeneration of shoots at high frequencies from mature cotyledon tissues of three genotypes of sunflower (Helianthus annuus L.) one of which had previously been found to be recalcitrant to regeneration when cotyledons were cultured on solid medium. Cotyledons were excised from 2-day-old seedlings and incubated in liquid Murashige and Skoog's modified medium supplemented with 5.4 M naphthaleneacetic acid (NAA) and 4.4 M benzylaminopurine (BAP). After two weeks in culture, the whole upper surface of regenerating explants was covered with green shootlets. The percentages of regenerating explants of three genotypes varied between 60 and 70%, and the number of shoots per regenerating explant was highly increased. The shootlets were transferred to solid Murashige and Skoog's medium allowing shoot development, then to rooting medium. Rooted plantlets were successfully acclimatized and gave fertile plants. The role of liquid medium culture in the induction of sunflower regeneration is discussed.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Leukoregulin, a T-cell derived cytokine, upregulates stromelysin-1 gene expression in human dermal fibroblasts: evidence for the role of AP-1 in transcriptional activation.

Leukoregulin (LR), a product of activated T-cells, has been recently shown to modulate the metabolism of extracellular matrix components in human skin fibroblast cultures (Mauviel et al., J Cell Biol 113:1455-1462, 1991). In this study we focused our attention on the effects of LR on the expression of stromelysin-1 gene. This matrix metalloprotease has a broad spectrum of degradative activity and it is also required for maximal activation of interstitial collagenase. Incubation of skin fibroblast cultures with LR resulted in a dose- and time-dependent elevation of stromelysin-1 mRNA levels, the maximum enhancement being up to approximately sevenfold. This effect was abolished by cycloheximide, suggesting a requirement for ongoing protein synthesis. Transient cell transfections with a promoter/reporter gene construct containing 1.3 kb of 5' flanking DNA of the human stromelysin-1 gene linked to the chloramphenicol acetyl transferase (CAT) gene, indicated enhancement of promoter activity by LR. This enhancement was abolished by a single base substitution in the AP-1 binding site of the promoter. Furthermore, gel mobility shift assays demonstrated enhanced AP-1 binding activity in nuclear extracts from cells incubated with LR. However, LR did not alter the activity of a construct containing three AP-1 sequences in front of the thymidine kinase promoter linked to the CAT gene. These results collectively suggest that activation of stromelysin-1 gene expression by LR is mediated by AP-1 regulatory elements which are necessary, but not sufficient, for gene response.     

Studies on NK cell purification by negative selection in human peripheral blood

We have attempted to improve negative selection procedures for the large scale purification of human CD _in3 ^– CD56⁺ NK cells. In a series of experiments, purifications of NK cells from 10⁸ PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3⁺ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10⁸ cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10⁹ PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.     

Parenteral supplementation with zinc in surgical patients corrects postoperative serum-zinc drop

Zinc has been known for a long time to facilitate wound healing. But, so far, supplementation trials in patients treated by major severity surgery gave either partial or controversial results. In a double-blind, randomized study including 30 patients, we show that zinc supplements (30 mg/d for 3 d) administered by a drip correct postoperative drop of serum zinc, that this correction concerns the available part of serum zinc (i.e., zinc that is bound to compounds other than alpha-2 macroglobulin in serum), and that this supplementation can improve clinical wound healing. Possible influence of increased urinary output after the intervention is discussed, and we found that serum cortisol remains stable when zinc/albumin ratio is stable, and increases sharply when the same ratio drops. Cortisol, therefore, seems to play a major role in zinc redistribution after surgery.     

Consumption by semen loss in India and elsewhere

Using ethnographical data from a rural site of Oriya culture, the purpose of this article is to show that the Indian obsession regarding the harmful consequences of semen loss does not constitute a specific Indian culture-bound preoccupation, but represents a survival example of a much widespread system of beliefs that can be traced for instance in Western culture to the Hippocratic notion of back consumption and the classical concept of gonorrhoea. At the core of this system of beliefs are found ancient conceptions of the nature and functions of semen in human life. These conceptions and their cultural influences incidentally inform us about one of the origins of the reprobation of onanism, as well as one possible way, among many others, for traditional thinking to explain the clinical enigma of depressive syndrome.     

d-Hydantoinase from anaerobic microorganisms

Summary Of 373 anaerobic microbial isolates screened for the enzymatic conversion of dihydrouracil to N-carbamyl--alanine, several strains of Clostridium spp., C. glycolicum, C. subterminale and Peptococcus anaerobius were positive. These Clostridium and Peptococcus strains produced also N-carbamyl-d-amino acids from the respective 5-monosubstituted hydantoins. The d-hydantoinase activity from whole cell suspensions of P. anaerobius strain CRDA 303 was characterized with regard to pH and temperature stability and activity by using dihydrouracil (DHU) and isopropylhydantoin (IPH) as substrates. The d-hydantoinase from P. anaerobius was optimal at 60°C and at pH 6.5–9.5 for the substrate DHU. It was stable up to 55°C and at pH 5.0–9.5 and could be stored at 4°C under an aerobic atmosphere for at least 14 days. Offprint requests to: A. Morin