Inhibition of Nb2 T-lymphoma cell growth by transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) inhibits proliferation of Nb2 cells, a rat T lymphoma, in response to lactogens and interleukin-2. Prostaglandins may play an important role in the pathway through which TGF-beta exerts its inhibitory actions, because prostaglandin E2 also inhibits proliferation of Nb2 cells, and indomethacin, an inhibitor of prostaglandin synthesis, reverses the inhibitory effects of TGF-beta on Nb2 cell proliferation.

Direct determination of activities for microorganisms of chesapeake bay populations.

We used three methods in determination of the metabolically active individual microorganisms for Chesapeake Bay surface and near-bottom populations over a period of a year. Synthetically active bacteria were recognized as enlarged cells in samples amended with nalidixic acid and yeast extract and incubated for 6 h. Microorganisms with active electron transport systems were identified by the reduction of a tetrazolium salt electron acceptor. Microorganisms active in uptake of amino acids, thymidine, and acetate were determined by microautoradiography. In conjunction with enumeration of active organisms, a total direct count was made for each sample preparation by epifluorescence microscopy. For the majority of samples, numbers of amino acid uptake-active organisms were greater than numbers of organisms determined to be active by other direct measurements. Within a sample, the numbers of uptake-active organisms (amino acids or thymidine) and electron transport system-active organisms were significantly different for 68% of the samples. Numbers of synthetically active bacteria were generally less than numbers determined by the other direct activity measurements. The distribution of total counts in the 11 samplings showed a seasonal pattern, with significant dependence on in situ water temperature, increasing from March to September and then decreasing through February. Synthetically active bacteria and amino acid uptake-active organisms showed a significant dependence on in situ temperature, independent of the function of temperature on total counts. Numbers of active organisms determined by at least one of the methods used exceeded 25% of the total population of all samplings, and from June through September, >85% of the total population was found to be active by at least one direct activity measurement. Thus, active rather than dormant organisms compose a major portion of the microbial population in this region of Chesapeake Bay.

Inhibitory effect of solar radiation on amino Acid uptake in chesapeake bay bacteria.

The effect of solar radiation on a natural bacterial population from the Chesapeake Bay was evaluated from measured changes in numbers of organisms engaged in amino acid uptake. From July through May, freshly collected water samples were exposed in quartz containers to 3.5 h of total sunlight both with and without UV-absorbing filters. Water samples were subsequently incubated with tritiated amino acids, and the uptake-active bacteria were assayed by microauto-radiography-epifluorescence microscopy. The survival index, defined as the fraction of the uptake-active population that remained active after the exposure to sunlight, ranged from 0.93 to 0.20. Decreased survival was correlated with increased solar intensity. The inhibition of amino acid uptake was attributed not only to the UV-B component of the solar spectrum (280 to 320 nm), but also to longer UV and visible wavelengths.

Improved microautoradiographic method to determine individual microorganisms active in substrate uptake in natural waters.

We report a method which combines epifluorescence microscopy and microautoradiography to determine both the total number of microorganisms in natural water populations and those individual organisms active in the uptake of specific substrates. After incubation with H-labeled substrate, the sample is filtered and, while still on the filter, mounted directly in a film of autoradiographic emulsion on a microscope slide. The microautoradiogram is processed and stained with acridine orange, and, subsequently, the filter is removed before microscopic observation. This novel preparation resulted in increased accuracy in direct counts made from the autoradiogram, improved sensitivity in the recognition of uptake-active (H-labeled) organisms, and enumeration of a significantly greater number of labeled organisms compared with corresponding samples prepared by a previously reported method.

Activity and growth of microbial populations in pressurized deep-sea sediment and animal gut samples.

Benthic animals and sediment samples were collected at deep-sea stations in the northwest (3,600-m depth) and southeast (4,300- and 5200-m depths) Atlantic Ocean. Utilization rates of [14C]glutamate (0.67 to 0.74 nmol) in sediment suspensions incubated at in situ temperatures and pressures (3 to 5 degrees C and 360, 430, or 520 atmospheres) were relatively slow, ranging from 0.09 to 0.39 nmol g-1 day-1, whereas rates for pressurized samples of gut suspensions varied widely, ranging from no detectable activity to a rapid rate of 986 nmol g-1 day-1. Gut flora from a holothurian specimen and a fish demonstrated rapid, barophilic substrate utilization, based on relative rates calculated for pressurized samples and samples held at 1 atm (101.325 kPa). Substrate utilization by microbial populations in several sediment samples was not inhibited by in situ pressure. Deep-sea pressures did not restrict growth, measured as doubling time, of culturable bacteria present in a northwest Atlantic sediment sample and in a gut suspension prepared from an abyssal scavenging amphipod. From the results of this study, it was concluded that microbial populations in benthic environments can demonstrate significant metabolic activity under deep-ocean conditions of temperature and pressure. Furthermore, rates of microbial activity in the guts of benthic macrofauna are potentially more rapid than in surrounding deep-sea sediments.

Improved method for determination of respiring individual microorganisms in natural waters.

A method is reported that combines the microscopic determinations of specific, individual, respiring microorganisms by the detection of electron transport system activity and the total number of organisms of an estuarine population by epifluorescence microscopy. An active cellular electron transport system specifically reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, which is recognized as opaque intracellular deposits in microorganisms stained with acridine orange. In a comparison of previously described sample preparation techniques, a loss of >70% of the counts of INT-reducing microorganisms was shown to be due to the dissolution of INT-formazan deposits by immersion oil (used in microscopy). In addition, significantly fewer fluorescing microorganisms and INT-formazan deposits, both

A pressure-retaining deep ocean sampler and transfer system for measurement of microbial activity in the deep sea.

A deep ocean sampler (DOS) has been developed for microbiological sampling and is capable of aseptically collecting 400-ml water samples from any depth in the world oceans. The instrument maintains samples under in situ pressure and temperature. A hyperbaric transfer system has also been developed, enabling transfer of sample volumes up to 150 ml, without decompression or dilution, to pressurized incubation chambers. Utilization of(14)C-glutamate (21 to 96µg/l) and(14)C-acetate (4.6µg/l) by microbial populations in undecompressed water samples from the N.W. Atlantic and the Cape and Angola Basins was recorded over incubation periods of 2 to 18 weeks. Rates of substrate utilization ranged from 1 to 38×10(-2) µg/l/day.

Filterable marine bacteria found in the deep sea: Distribution, taxonomy, and response to starvation.

A significant number of viable colony-forming bacteria were recovered from deep-ocean bottom water samples passed through a 0.45µm filter. However, these bacteria small enough to pass through a 0.45µm membrane filter and termed "filterable bacteria" were less abundant in open-ocean surface water and coastal water samples. The reduced size of bacterial cells present in deep-ocean bottom water samples was documented by scanning electron microscopy. The concentration of ATP in the water samples was found to be correlated with results of direct counts of bacteria.Numerical taxonomy of bacterial strains isolated from water samples collected at two stations in the deep sea yielded taxonomic clusters grouped according to sample and size fraction. The generic composition of bacterial populations of bottom water filtrates was compared with that of bacteria retained by 0.45µ m filters. Strains ofAlcaligenes, Flavobacterium, Pseudomonas, andVibrio spp. were identified among those retained by, as well as passing through, 0.45µm filters.Two marine isolates obtained from the filtrate of a deep-ocean water sample were incubated for 9 weeks in nutrient-free artificial seawater, during which the cells became rounded and reduced in size. After the 9-week incubation period, more than 10% of the viable cells of both cultures were able to pass through a 0.4µm filter. The viable count at 9 weeks wasca. 10% of that of the initial population, although from direct counts the total population number remained relatively constant throughout the incubation period. From the observed reduction in cell size and increased starvation resistance of cells held under low nutrient conditions, it is concluded that a significant relationship exists between decreased cell size and increased survival of marine bacteria in the deep sea.

Barophilic growth of bacteria from intestinal tracts of deep-sea invertebrates.

Digestive tracts of abyssal scavenging amphipods and a deep-sea holothurian were examined for the presence of intestinal microflora capable of rapid proliferation under in situ pressures of 430 to 520 atmospheres (atm) and temperatures of 3-5°C. For two amphipod specimens, population doubling times of 5 and 6 hours were observed under in situ conditions, compared to 8 and 6 hours, respectively, at 1 atm. Growth enhancement under pressure was related inversely to initial population size and directly to concentration of available nutrient. In the case of the deposit-feeding holothurian, attached bacteria scraped from the intestinal lining showed a doubling time, under pressure, of 11 hours, compared to 36 hours for transient sediment bacteria that comprised the gut contents. These data suggest that deep-sea animals possess a commensal gut flora capable of responding to increased nutrient levels, via feeding of the host, without inhibition by the elevated hydrostatic pressures encountered in the deep ocean environment.

Species composition and barotolerance of gut microflora of deep-sea benthic macrofauna collected at various depths in the atlantic ocean.

The bacterial flora of marine animals collected at depths of 570 to 2,446 m was examined for population size and generic composition, and the barotolerant characteristics of selected bacterial isolates were determined. Total numbers of culturable, aerobic, heterotrophic bacteria were found to be low in animals collected at the greatest ocean depths sampled in this study. Vibrio spp. were predominant in 10 of 15 samples examined, and Photobacterium spp. and yeasts were the major components of the remainder. Pseudomonas, Achromobacter, and Flavobacterium spp. comprised minor components of the gut flora of deep-sea fish. Forty-six pure cultures isolated from samples of seven animals were tested for growth or viability after incubation for 1 week under pressures ranging from 100 to 750 atm. Strains of bacteria isolated from samples of fish intestine were more barotolerant than those from the stomach (P<0.01). When incubated at a pressure of 600 atm, viability of bacterial cultures originally isolated from fish caught at a depth of 570 m was significantly decreased in comparison with viability of cultures from animals caught at depths of 1,393 and 2,446 m (P<0.01). From results of this study, it is concluded that the gut microflora of animals that dwell in the deeper regions of the ocean are adapted to an increased hydrostatic pressure environment, that is, the gut microflora is less inhibited by elevated hydrostatic pressure with increasing depth from which the host animal was collected.