Lectin binding as a probe of proliferative and differentiative phases in primary monolayer cultures of cutaneous keratinocytes

The surface of cells in the cutaneous epidermis of the newborn rat exhibits a discrete change in lectin-binding specificity from Griffonia simplicifolia I-B4 (GS I-B4), specific for alpha-D-galactosyl residues, to Ulex europeus agglutinin I (UEA), specific for alpha-L-fucose, as the cell leaves the basal layer and differentiates. Primary monolayer cultures of rat keratinocytes maintained in low Ca2+ medium (0.08 mM) exhibited a characteristic unimodal pattern in the ratio of bound UEA to bound GS I-B4 (UEA/B4 ratio) over a 7-day culture period as determined by a quantitative fluorometric assay. The UEA/B4 ratio was initially low between Days 1 and 2 (0.56 +/- 0.05), steadily increased to a maximum of 0.84 +/- 0.09 between Days 2 and 4, and then gradually decreased to 0.41 +/- 0.07 between Days 6 and 7. Estimation of DNA synthesis showed (a) a higher [3H]thymidine incorporation when the UEA/B4 ratio was low and (b) a steady but lower incorporation between Days 3 and 4, coincident with the higher UEA/B4 ratio. Autoradiographic results further showed that cells stained intensely with UEA failed to incorporate [3H]thymidine into their nuclei. Electrophoresis of [3H]fucose-labeled material isolated on UEA-Sepharose 4B revealed that the changes in labeling by [3H]fucose, bound UEA, and the UEA/B4 ratio in the monolayer were related in part to variable expression of "96K-associated UEA-bound" radioactivity corresponding to a major class of lectin-specific cell-surface glycoproteins (GP96 fraction) identified in situ. Overall, the results suggest that (a) the increase in the UEA/B4 ratio between Days 2 and 4 reflects the progression of a proportion of the cells in the monolayer to an early spinous cell stage, the ultimate fate of which is desquamation into the medium and (b) the decrease in the UEA/B4 ratio between Days 5 and 7 reflects a consequent proliferative response to this loss of cells. This system should be useful for studying environmental influences on the homeostasis of cell proliferation and differentiation in the cutaneous epidermis.     

Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase

The dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was specifically labelled with 13C by growth of the bacteria in the presence of 5-amino[5-13C]levulinic acid. Using 13C-NMR spectroscopy, the structure of the cofactor was confirmed as a dipyrromethane made up of two linked pyrrole rings each derived from porphobilinogen. The chemical shift data indicate that one of the pyrrole rings of the cofactor is covalently linked to the deaminase enzyme through a cysteine residue. Evidence from protein chemistry studies suggest that cysteine-242 is the covalent binding site for the cofactor.     

Structure and transcription analysis of the gene encoding a cellobiase from Agrobacterium sp. strain ATCC 21400

The DNA sequence was determined for the cloned Agrobacterium sp. strain ATCC 21400 beta-glucosidase gene, abg. High-resolution nuclease S1 protection studies were used to map the abg mRNA 5' and 3' termini. A putative abg promoter was identified whose sequence shows similarities to the consensus promoter of Escherichia coli and with the nif promoter regions of Klebsiella. The abg coding sequence was 1,374 nucleotides long. The molecular weight of the enzyme, based on the predicted amino acid sequence, was 51,000. The observed Mr was 50,000 to 52,000. A region of deduced protein sequence was homologous to a region from two other beta-glucosidase sequences. This region of homology contained a putative active site by analogy with the active site of hen egg white lysozyme.     

The effects of fetal exposure to 1,2-dibromo-3-chloropropane on adult male reproductive function

Several drugs have been shown to cross the placental barrier and affect the fetal testis causing a reduction in testosterone with a resultant impairment of sexual differentiation and an ultimate problem in adult sexual function. In this study, pregnant female rats were treated with 25 mg/kg of the pesticide 1,2-dibromo-3-chloropropane (DBCP). Treatment began on Days 14.5, 16.5, or 18.5 and continued through Day 19.5 of gestation. Some animals were killed on Day 20.5 of intrauterine life and fetal intratesticular testosterone was measured. All other animals were allowed to deliver, and the males were raised to adulthood. At adulthood, body, testis, prostate glands and seminal vesicle weights were recorded. Intratesticular testosterone and luteinizing hormone (LH) receptors were measured. Male and female sexual behavior was quantified and the volume of the sexually dimorphic nucleus of the preoptic area of the hypothalamus was calculated. The histological appearance of the testis was also examined. Treatment for 6 days during fetal life with DBCP decreased intratesticular testosterone by 50% compared to controls at 20.5 days of gestation. At adulthood, all male rats treated during fetal life had a reduced body weight that was correlated with the duration of exposure. Adult testis weight was reduced to 75% of controls as a result of 2 days of fetal exposure to DBCP, whereas 4 and 6 days of exposure during fetal life reduced testis weight by greater than 90%. LH receptors and intratesticular testosterone, in the adults treated during fetal life, were also dramatically reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Macrophage growth factor CSF-1 stimulates human monocyte production of interferon, tumor necrosis factor, and colony stimulating activity

CSF-1, a macrophage colony stimulating factor that causes proliferation and differentiation of progenitor cells, may also have effects on mature cells. Human peripheral blood monocytes were used to examine this possibility. Monocytes, separated from normal blood by density centrifugation and adherence, were incubated for 3 days with or without CSF-1 (1,000 U/ml, purified from the MIA PaCa pancreatic carcinoma line). The two groups of cells were then washed and tested for the ability, when induced, to produce several factors. When induced for 2 days with LPS and PMA, the monocytes produced a factor that was cytotoxic to L929 cells, and this factor was completely neutralized by polyclonal antibody to tumor necrosis factor. The cells preincubated with CSF-1 consistently produced an average of 12 times more of this factor than cells not exposed to CSF-1. Monocytes induced with LPS and PMA also produced a colony stimulating activity, as measured by colony formation when using mouse bone marrow. Cells preincubated with CSF-1, washed, and induced with LPS and PMA produced more than three times as much activity compared with control monocytes. When monocytes were induced with poly-I.C, 22-fold higher levels of interferon were produced by the cells exposed to CSF-1. These results show that CSF-1 has direct stimulating effects on mature human monocytes, and suggest that the macrophage growth factor may have clinical application in the treatment of infectious diseases and cancer.     

Nutrient and biomass allocation in Solidago altissima: effects of two stem gallmakers,fertilization, and ramet isolation

Summary The allocations of biomass, N, P, and K were determined by standard methods in goldenrod ramets (1) parasitized by dipteran and lepidopterous gallmakers, (2) from fertilized and unfertilized plots, and (3) whose rhizome connections to their parental clone were severed. The presence of ball galls and their larvae increased allocation to stem but decreased allocation to leaves and seed production, and reduced the number of new rhizomes. There was a marked magnification of N and P concentrations going up the food chains; from goldenrods to gallmakers to the gallmaker's parasitoid/inquiline guild. Nutrient budgets expressed as flow diagrams indicated that N and P costs of gall presence were similar to energy costs under conditions where nutrients did not limit plant growth. Our data do not indicate that the growth of the galls of these gallmakers is limited by either N or P. Ramets from fertilized plots contained more N and P than controls but decreased the percentage of biomass allocated to leaves and inflorescences; ramets isolated by rhizome-cutting compensated their loss by increased allocation to roots, current rhizomes, and new rhizomes but at a cost of lower allocation to seed production and leaves. Gallmakers have a negative impact on host plant fitness characteristics. This may be especially important to establishing perennial hosts, given that herbivore effects would reduce clonal expansion and hence the ultimate clone size, thereby decreasing lifetime plant fitness.     

Bone marrow-derived macrophages: development and regulation of differentiation markers by colony-stimulating factor and interferons

To investigate the role of specific cytokines in the development of the fully mature macrophage, we have employed murine bone marrow cells that were grown in the presence of CSF-1, a colony-stimulating factor that has been shown to induce the proliferation and differentiation of macrophages from their precursor cells. The CSF-1 employed in these studies was partially purified to ensure removal of contaminating interferon (IFN) from the preparations. After 1 to 2 wk in the presence of the partially purified CSF-1, the adherent macrophages were removed from flasks enzymatically and were recultured at known densities in the absence of CSF-1. Cell surface antigens (Mac-1 and Ia) and Fc receptor capacity (as assessed by Fc-mediated phagocytosis) were examined as markers of macrophage differentiation. Basal levels of Fc receptor capacity and Mac-1 antigen were markedly influenced by exposure to CSF-1, and appear to be modulated by CSF-induced, macrophage-derived IFN. When the bone marrow-derived macrophages were exposed to exogenous IFN in the absence of CSF-1, they proved to be extremely inducible with respect to Fc-mediated phagocytosis (IFN-beta and rIFN-gamma) and Ia antigen expression (rIFN-gamma) when compared with thioglycollate-elicited macrophages. Thus, macrophage growth factors, such as CSF-1, promote macrophage maturation by inducing the production of autostimulatory signals, such as macrophage-derived IFN. In addition, exogenous cytokine stimuli, such as IFN-gamma, further amplify the differentiative potential of these cells. Bone marrow-derived macrophages, propagated under well-defined conditions and never exposed to eliciting agents, provide a powerful model for studying the role of cytokines, such as CSF-1 and IFN, in the differentiative pathway of macrophages.