Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex

The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme.     

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.     

Studies of comparative infectivity of fifteen strains of Plasmodium vivax to laboratory-reared anopheline mosquitoes, with special reference to Anopheles culicifacies

The Sattoki strain of species A of the taxon Anopheles culicifacies Giles was infected with 15 different strains of Plasmodium vivax from Asia, New Guinea, and Central and South America. A comparison of the relative infectivity indicated a marked variation for the different strains of P. vivax when compared to Anopheles freeborni mosquitoes.     

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.     

Antagonistic effect of interferon-beta on the interferon-gamma-induced expression of Ia antigen in murine macrophages

The cell surface expression of I region-associated (Ia) antigens by murine and human macrophages has been shown by investigators from a number of laboratories to be induced in a dose-dependent fashion by IFN-gamma, which is free of other lymphokines. The experiments described in this report demonstrate that fibroblast-derived IFN-beta exerts an antagonistic effect on IFN-gamma induced Ia expression in murine macrophages. Simultaneous addition of IFN-beta and IFN-gamma to peritoneal exudate macrophages results in decreased Ia expression when compared with macrophages treated with IFN-gamma only. Different sources of highly purified IFN-beta, as well as a recombinant human IFN-alpha (A/D Bgl; shown previously to be as active as IFN-beta in several other murine systems) acted in a similar antagonistic fashion to IFN-gamma-induced Ia induction. The down-regulation of Ia expression by IFN-beta is dose-dependent over a concentration range up to 100 U/ml. Time-course experiments indicated that for IFN-beta to down-regulate IFN-gamma-induced Ia, it had to be present either before stimulation with IFN-gamma or during the first 24 hr of simultaneous stimulation. Further experiments in which a highly specific antibody against IFN-alpha/beta was added to the cultures confirmed the findings of the time-course experiments. Inhibitors of the arachidonic acid pathway failed to reverse the effect of IFN-beta to reduce Ia antigen expression, which suggests that this inhibition is not prostaglandin mediated. Thus, these findings support a role for type I IFN as naturally occurring substances that negatively regulate the expression of class II molecules.     

Evolutionary impacts of the goldenrod ball gallmaker on Solidago altissima clones

Summary Three ramet clones of Solidago altissima were grown under greenhouse conditions to determine the effects of varying levels of attack by the goldenrod ball gallmaker (Eurosta solidaginis) on biomass allocation, leaf senescence rate and rhizome connections among ramets. The results, examined at both the individual ramet level and the clone level, showed that galled ramets became isolated from their clone through deterioration of rhizome connections. Gall effects were only observed at the ramet level although rhizome connection effects were detected at both the ramet and clone levels. The goldenrod ball gallmaker may therefore have little evolutionary impact on large clones but could appreciably affect newly established clones.     

Purification and characterization of a low molecular weight transforming growth factor from the urine of melanoma patients

A low Mr human transforming growth factor (TGF) present in melanoma patients' urine has been purified approximately 200,000-fold to apparent homogeneity. Initial purification of an acid-soluble fraction of urine was achieved by Bio-Gel P-30 gel filtration chromatography in 1 M acetic acid. TGF activities were demonstrated in the Mr ranges of 30,000 and 6,000-10,000. These competed with epidermal growth factor (EGF) for binding to A431 membrane receptors and induced anchorage-independent growth of untransformed fibroblasts. The low Mr TGF activity obtained from P-30 chromatography was purified to apparent homogeneity by two sequential reverse-phase high performance liquid chromatography steps with a mu Bondapak C18 column first using a linear gradient of acetonitrile going from 0-60% in 120 min and then by rechromatography of the activity over the same column using a shallower gradient of acetonitrile going from 20-40% in 160 min. The isoelectric point of the melanoma patient-derived urinary TGF was determined to be 6.2, which is distinct from that for human EGF. Amino acid composition analysis of the purified urinary TGF (uTGF) revealed that it is composed of at least 42 amino acid residues with a minimum estimated Mr of 4,545. Compositional analysis further revealed distinct similarities and differences between the uTGF, human EGF and TGFs secreted by various transformed human and rodent cell lines.     

Mechanism of transient nocturnal hypoxemia in hypoxic chronic bronchitis and emphysema

In five patients with hypoxic chronic bronchitis and emphysema we measured ear O2 saturation (SaO2), chest movement, oronasal airflow, arterial and mixed venous gas tensions, and cardiac output during nine hypoxemic episodes (HE; SaO2 falls greater than 10%) in rapid-eye-movement (REM) sleep and during preceding periods of stable oxygenation in non-REM sleep. All nine HE occurred with recurrent short episodes of reduced chest movement, none with sleep apnea. The arterial PO2 (PaO2) fell by 6.0 +/- 1.9 (SD) Torr during the HE (P less than 0.01), but mean arterial PCO2 (PaCO2) rose by only 1.4 +/- 2.4 Torr (P greater than 0.4). The arteriovenous O2 content difference fell by 0.64 +/- 0.43 ml/100 ml of blood during the HE (P less than 0.05), but there was no significant change in cardiac output. Changes observed in PaO2 and PaCO2 during HE were similar to those in four normal subjects during 90 s of voluntary hypoventilation, when PaO2 fell by 12.3 +/- 5.6 Torr (P less than 0.05), but mean PaCO2 rose by only 2.8 +/- 2.1 Torr (P greater than 0.4). We suggest that the transient hypoxemia which occurs during REM sleep in patients with chronic bronchitis and emphysema could be explained by hypoventilation during REM sleep but that the importance of changes in distribution of ventilation-perfusion ratios cannot be assessed by presently available techniques.