Virulence genes are carried by a megaplasmid of the plant pathogen Pseudomonas solanacearum

Summary A class of avirulent mutants of the plant pathogenic bacterium Pseudomonas solanacearum, strain GMI1000, resistant to acridine orange (Acr^r), harbour a deletion of over 85 kb in their genome. This deletion affects, a1,000 kb megaplasmid which has previously been shown to be present in most of the strains of this species. In addition at least 11 out of 13 independent Tn5 insertions, leading to loss of virulence, are located on the megaplasmid. Nine of them are present in the region which is deleted from the Acr^r mutants. These results suggest that the majority of virulence genes identified so far are plasmid borne.     

Identification of host range determinants in the Rhizobium species MPIK3030

Summary R. meliloti primarily nodulates Medicago sativa but cannot nodulate Macroptilium atropurpureum. By introducing an 11.4 kb region into R. meliloti from the Symplasmid of Rhizobium strain MPIK3030, the host range of the R. meliloti transconjugants were shown to be extended to M. atropurpureum, one of the hosts of MPIK3030 but not normally nodulated by R. meliloti. The region responsible for host range extension was isolated by mass conjugating a clone bank from MPIK3030 into the R. meliloti wild type, and subsequent screening for nodulation on M. atropurpureum. Using deleted derivatives of a plasmid reisolated from endosymbiotic bacteria, the host range region was further narrowed down to three EcoRI fragments. Tn5 mutagenesis allowed the isolation of three discrete regions on an 11.4 kb section, which are involved in the extension of host range to M. atropurpureum. Finally, complementation experiments performed with R. meliloti common nod and hsn mutants indicated that none of the genes involved in the early steps of nodulation, including host-range functions, can be complemented by genes carried on the 11.4 kb fragment derived from MPIK3030.     

Regulation of major acute-phase plasma proteins by hepatocyte- stimulating factors of human squamous carcinoma cells

Human squamous carcinoma (COLO-16) cells release factors which specifically stimulate the synthesis of major acute-phase plasma proteins in human and rodent hepatic cells. Anion exchange, hydroxyapatite, lectin, and gel chromatography of conditioned medium of COLO-16 cells result in separation into three distinct forms of hepatocyte-stimulating factors (designated HSF-I, HSF-II, and HSF-III) with apparent molecular weights of 30,000, 50,000 and 70,000, respectively. None of the preparations contains detectable amounts of thymocyte-stimulating activity. Each of the three HSF forms stimulates the accumulation of mRNA for alpha 1-antichymotrypsin in the human hepatoma cell line, HepG2. When the same factors were added to primary cultures of adult rat hepatocytes, the expression of the same set of plasma proteins was modulated as by nonfractionated medium. The hormonally induced accumulation of mRNA for acute phase proteins is qualitatively comparable to that occurring in the liver of inflamed rats. Unlike in human cells, in rat liver cells dexamethasone acts additively and synergistically with HSFs. The only functional difference between the three HSF forms lies in the level of maximal stimulation. HSF-I represents the predominant form produced by normal human keratinocytes and closely resembles in molecular size and isoelectric point the activity produced by activated peripheral blood monocytes while the larger molecular weight forms are more prevalent in human as well as mouse squamous carcinoma cells. The observation that HSFs from different sources elicit essentially the same pleiotropic response in hepatic cells led to the hypothesis that the species-specific reaction of adult liver cells to inflammatory stimuli is pre-programmed and that the function of any HSF is to trigger and tune the execution of this fixed cellular process.     

Polyester Wadding for Specimen Orientation During Embedding in Methacrylates

Polyester fibers are not dissolved by either glycol methacrylate or methyl methacrylate. Commercial polyester wadding is consequently an advantageous material to use in getting precise orientation of tissue specimens during embedding in methacrylate.     

Permanent Preservation of Whole Alizarin Red S Skeletons by Clearing and Embedding in Polyester Resins

A one-step clearing and embedding procedure for alizarin red S stained skeletons is described. Embryos are fixed in formalin, skinned and eviscerated. After staining in a 10 mg/liter solution of alizarin red S in 5% aqueous KOH, specimens are dehydrated in a graded series of acetone-polyester monomer solutions. Finally, the specimens are embedded at room temperature in the polyester resin. A special reusable metallic mold is described for embedment of large fetuses. Specimens previously cleared in glycerol can be processed with this method.     

Metabolism and Solubilization of Cellulose by Clostridium cellulolyticum H10

When Clostridium cellulolyticum was grown with cellulose MN300 as the substrate, the rates of growth and metabolite production were found to be lower than those observed with soluble sugars as the substrate. At low cellulose concentrations, the growth yields were equal to those obtained with cellobiose. The main fermentation products from cellulose and soluble sugars were the same. Up to 15 mM of consumed hexose, a change in the metabolic pathway favoring lactate production similar to that observed with soluble sugars was found to occur concomitantly with a decrease in molar growth yield. With cellulose concentrations above 5 g/liter, accumulation of soluble sugars occurred once growth had ceased. Glucose accounted for 30% of these sugars. A kinetic analysis of cellulose solubilization revealed that cellulolysis by C. cellulolyticum involved three stages whatever cellulose concentration was used. Analysis of these kinetics showed three consecutive enzymatic activity levels having the same K_m (0.8 g of cellulose per liter, i.e., 5 mM hexose equivalent) but decreasing values of V_max. The hypothesis is suggested that each step corresponds to differences in cellulose structure.     

Genetics and evolution of the acute phase proteins in mice

Summary In mammals, a number of liver-derived plasma proteins, termed acute phase reactants, are induced during an inflammation response. We have studied genetic variation in the structure and expression of several of these proteins in a variety of inbred and wild-derived mice. In a genetic cross, electrophoretic polymorphisms for the two ₁-acid glycoproteins, AGP-1 and AGP-2, co-segregated in 58 backcross progeny, indicating that either a single gene or two tightly-linked genes on chromosome 4 encode the AGPs. In the same backcross, segregation of variation in haptoglobin structure showed that the gene encoding this acute phase reactant is on chromosome 8. Structural variation in serum amyloid A correlated with restriction fragment length polymorphisms in the Saa gene determined by Taylor and Rowe (1984). Analysis of a number of highly diverged species of mice indicated that AGP expression has undergone considerable modification during evolution of the Mus genus; this is associated with alterations in Agp gene organization, which may include species-specific amplification and/or deletion events.     

Physical and biotic determinants of space utilization by the Galapagos land iguana (Conolophus pallidus)

Summary Home ranges of the Galapagos land iguana (Conolophus pallidus) were examined with respect to food availability and the thermal environment. Activity patterns, the amount of space used per day, and time required to use the entire home range were also investigated. The effects of, and the relationships between, these factors vary seasonally, as do home range sizes and preferred body temperatures.Food supplementation experiments resulted in only temporary reductions in use of space. Home range sizes were not different between the seasons with the least (Fall) and the most (Hot) food availalble, but home ranges were significantly smaller in Garua when food supplies were low, but not as low as in Fall. Calculations of metabolic expenditures in each season suggests that food availability alone does not explain seasonal patterns of home range size in this species.The thermal environment within each home range was characterized by microclimatic measurements and measurements of the area of sun, shade, and semi-shade. An index with units of m²h was used to quantify the thermal quality of each home range. Iguanas exploited optimal (with respect to body temperature) conditions more than would be expected from random use of their home ranges. Thermal transients (due to large body size) and optimal conditions were exploited to the largest degree in Fall.During Garua, low metabolic rates and time constraints imposed by an abundance of stressful thermal environments may result in small home ranges. In Fall, increased temperatures cause higher metabolic rates and allow more time for exploitation of the cooler portions of the home range, hence, home range sizes increase. In the Hot season, there is abundant food and optimal thermal conditions, but home ranges remain large. Searching for preferred foods may cause the large home ranges in this season.     

Proliferative heterogeneity of vascular smooth muscle cells and its alteration by injury

This study was designed to explore the question of whether the population of morphologically similar smooth muscle cells (SMC) in the vessel wall is functionally homogeneous or heterogeneous with respect to their proliferative response to injury. Using time-lapse video recording we measured interdivision times (IDT) of primary SMC clones, sibling pairs, and mother/daughter pairs. SMC from in vivo undisturbed vessels displayed an interclonal and intraclonal heterogeneity with a wide range in IDT. In vivo balloon injury resulted in a population with homogeneously short IDT. While 80% of IDT of SMC from injured vessels were shorter than 14 h, only slightly more than half of IDT of cells from undisturbed vessels fell into this category. Longitudinal analysis of mother/daughter pairs confirmed the presence of a heterogeneous population of SMC in the undisturbed vessel wall. In vivo balloon injury not only shortened the IDT of the majority of cells, but the shorter IDT persisted much longer than in the case of the undisturbed vessel. We suggest that a morphologically homogeneous SMC population in the aorta can now be subdivided into several groups of functionally different SMC with respect to their proliferative response to injury.