A growth inhibitory protein secreted by human diploid fibroblasts. Partial purification and characterization

We have identified and partially purified a growth inhibitor protein secreted by human diploid fibroblast cells. This protein is not secreted constitutively but only after induction with the double stranded hetero duplex polyriboinosinic:polyribocytidylic acid. The growth inhibitory activity has been purified 3,800-fold and has an estimated molecular mass of 12,000 daltons. The protein will inhibit the growth in culture of human diploid fibroblast cells, human cells derived from tumors, and mouse L cells. Although interferon-beta is secreted with the growth inhibitory protein, the partially purified growth inhibitory protein has no antiviral activity, and its activity is not neutralized by antibodies to interferon-alpha, interferon-beta, and interferon-gamma. We believe this growth inhibitory activity to reside in a newly defined protein and have named it fibroblast-derived growth inhibitor.     

Temperature-Light Intensity Effect on the Antagonism of Species of Cladosporium to Melampsora larici-populina on Cultivars of Populus X euramericana (Dode) Guinier

The antagonistic and colonistic effect of post-inoculation temperature and light intensity was investigated in a factorial experiment involving four Cladosporium spp., three poplar cultivars, two levels of temperature and light intensity, anda race of Melampsora larici-populina. The species of Cladosporium were more antagonistic to uredinial production on the inoculated leaf discs of the cultivars when these were incubated in high temperature – high light intensity than in any other regime. Whilst colonisation of the uredinia was usually minimal in discs incubated on the high temperature – high light intensity regime, the condition of incubation favouring maximum colonisation varied with the cultivar and the species of Cladosporium employed. All the major factors and most of their two-, three- and four-way interactions were significant determinants of variation in both antagonism to uredinial production and in colonisation of the uredinia produced. The validity ofextrapolation from such experiments to the field environment is discussed.     

Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains

Summary A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD ^- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.     

Low temperature delays development of photosystem II activity in winter rye leaves

The ability of leaves to acclimate photosynthetically to low temperature was examined during leaf development in winter rye plants ( Secale cereale L. cv. Puma) grown at 20°C or at 6°C. All leaves grown at 6°C exhibit increased chlorophyll (Chl) levels per leaf area, higher rates of uncoupled, light-saturated photosystem I (PSI) electron transport, and slower increases in photosystem II (PSII) electron transport capacity, when compared with 20°C leaves. The stoiehiometry of PSI and PSII was estimated for each leaf age class by quantifying Chl in elcctrophorctic separations of Chl-protein complexes. The ratio of PSII/PSI electron transport in 20°C leaves is highly correlated with the ratio of core Chl a -proteins associated with PSII (CPa) to those associated with PSI (CP1). In contrast, PSII/PSI electron transport in 6°C leaves is not as well correlated with CPa/CP1 and is related, in part, to the amount and organization of light-harvesting Chl a/b -proteins associated with PSII. CPa/CP1 increases slowly in 6°C leaves, although the ratio of CPa/CP1 in mature 20°C and 6°C leaves is not different. The results suggest that increased PSI activity at low temperature is not related to an increase in the relative proportion of PSI and may reflect, instead, a regulatory change. Photosynthetic acclimation to low environmental temperature involves increased PSI activity in mature leaves shifted to 6°C. In leaves grown entirely at 6°C, however, acclimation includes both increased PSI activity and modifications in the rate of accumlation of PSII and in the organization of LHCII.     

Simple, rapid method for direct isolation of nucleic acids from aquatic environments

Direct isolation of nucleic acids from the environment may be useful in several respects, including the estimation of total biomass, detection of specific organisms and genes, estimations of species diversity, and cloning applications. We have developed a method that facilitates the concentration of microorganisms from aquatic samples and the extraction of their nucleic acids. Natural water samples of 350 to greater than 1,000 ml are concentrated on a single cylindrical filter membrane (type SVGS01015; Millipore Corp., Bedford, Mass.), and cell lysis and proteolysis are carried out within the filter housing. Crude, high-molecular-weight nucleic acid solutions are then drawn off the filter. These solutions can be immediately analyzed, concentrated, or purified, depending on the intended application. The method is simple, rapid, and economical and provides high-molecular-weight chromosomal DNA, plasmid DNA, and speciated RNAs which comigrate with 5S, 16S, and 23S rRNAs. The methods presented here should prove useful in studying both the ecology and the phylogeny of microbes that resist classical culture methods.     

Evidence for chromosomal replicons as units of sister chromatid exchanges

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 m with a peak value around 0.3 to 0.6 m regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments. A concentration of units around 0.4 to 1.0 m was found for L/S SCD segment size distributions regardless of CP treatment with an apparent bimodal profile. Our in vivo data support the existence of a subunit organization of chromosomal replication with a basic functional unit being 0.3 to 0.6 m in size. In addition, we have found that this chromosomal unit of replication or chromosomal replicon does not seem to be functionally perturbed by the mutagen CP. We also found that small SCD segments of 0.4 to 0.7 m in length were involved in the formation of an SCE, suggesting that both spontaneous and CP-induced SCEs occur between chromosomal replicons. These findings provide direct cytogenetic evidence to support a replicon cluster/chromosomal replicon model for SCE formation.     

The occurrence and distribution of certain polypeptides within the human carotid body

Summary Both carotid bodies from 26 patients coming to necropsy were fixed in 10% neutral buffered formalin and sections 4 m thick were stained for various peptides by use of the immunogold technique. The results show that the human carotid body contains met- and leu-enkephalin, substance P, vasoactive intestinal peptide (VIP), neurotensin and bombesin. The distribution of these six peptides within the carotid body differs. Thus met- and leu-enkephalin are both present predominantly within glomic chief cells but with a marked tendency to favour the dark variant of these cells. Substance P and VIP both show a weak immunoreactivity in comparison to the enkephalins and are present in all three variants of chief cell. Neurotensin shows the weakest immunoreactivity of all and is restricted to a few glomic chief cells in a minority of cases. Bombesin also shows a weak immunoreactivity in glomic chief cells but a strong reaction in glomic arteries and arterioles. In these vessels bombesin appears to be confined to smooth muscle cells in the media but we cannot say whether it is secreted by them or merely bound to receptor sites on their membranes. These findings are related to quantitative data on the concentration of peptides in the human carotid body from a previous paper with which we were associated.     

Screening for prolonged incubation of HTLV-I infection in British and Jamaican relatives of British patients with tropical spastic paraparesis.

OBJECTIVE--To compare the prevalence of antibody to and proviral DNA of the retrovirus HTLV-I in relatives of 11 British patients with tropical spastic paraparesis who had migrated from Jamaica before they developed symptoms, and to examine factors possibly related to transmission of HTLV-I. DESIGN--Migrant, family study. Antibody state was determined by several methods and confirmed by western blotting; the polymerase chain reaction was used to detect proviral DNA. SETTING--Britain and Jamaica. SUBJECTS--All available first degree relatives: those born and still resident in Jamaica (group 1); those born in Jamaica who migrated to Britain (group 2); and index patients'' children who were born and resident in Britain (group 3). All had been breast fed and none had had blood transfusions. RESULTS--Of the 66 living relatives, 60 were traced. Seroprevalence among those born in Jamaica (irrespective of current residence) was 22% (10/46; 95% confidence limits 9 to 34%) compared with zero among British born offspring (0/14) and was higher in group 2 at 33% (7/21; 12 to 55%) than in group 1 at 12% (3/25; 0 to 25%). (Patients in group 1 had the greatest mean age.) Proviral DNA was not detected in any subject negative for HTLV-I antibody, making prolonged viral incubation in those negative for the antibody unlikely. CONCLUSION--In this sample factors related to place of birth and early residence were more important in transmission of HTLV-I than maternal or age effects. In areas with a low to moderate prevalence policies of preventing mothers who are carriers of the virus from breast feeding would be premature.     

Minitron II system for precise control of the plant growth environment

A transparent, cylindrical chamber system was developed to allow measurement of gas-exchange by small crop canopies in the undisturbed plant growth environment. The system is an elaboration of the Minitron system developed previously to compare growth of small plants in different environments within the same general growth area. The Minitron II system described herein accommodates hydroponic culture and separate control of atmospheric composition in individual chambers. Root and shoot environments are compartmented separately to accommodate atmospheres of different flow rate and/or gaseous composition. A series of 0-rings and tension-adjustable springs allow carbon dioxide in the flowing atmosphere to be analyzed without cross-contamination between chamber compartments or from external gas sources. Carbon dioxide has been maintained at set point +/- 9 g m-3 over a range of CO2 concentrations from 382 to 2725 g m-3 and with an atmosphere turnover rate of 136.7 cm3 s-1 by computer-assisted mass flow controllers. Each chamber has dimensions large enough (61 cm internal diameter, 0.151 m3 internal volume) to allow adequate replication of individual plants for statistical purposes (e.g., up to 36 equally-spaced plant holders). No significant variation in growth or photosynthetic rate of leaf lettuce occurred between chambers for a given set of environmental conditions. Gas-exchange rates in different chambers changed to a similar extent as CO2 concentration in the flowing atmosphere or chamber temperature were varied by the same amount. When coupled with appropriate control systems, Minitron II chambers can provide separate controlled environments for multiple small plants with adequate precision and at relatively low cost.