Estimation Of Cell Number Research Articles

Alcohol exposure during the first two trimesters equivalent alters granule cell number and neurotrophin expression in the developing rat olfactory bulb.

Although alcohol has been shown to affect brain development adversely, the underlying mechanism of alcohol's actions are poorly understood. The present study addressed the hypothesis that alcohol affects growth factor availability during critical periods of neural growth by measuring the mRNA expression of brain-derived neurotrophic factor (BDNF), a potent developmental growth factor. Multiple offspring of timed-pregnant rat dams given alcohol (6.0 g/kg per day) or control treatments during gestation were sacrificed at either embryonic (E) day 21 or E33 (usually postnatal day 10) when their olfactory bulbs were processed for molecular analyses or neuron counting. BDNF mRNA levels were measured by reverse-transcription-polymerase chain reaction, and DNA methylation of the BDNF gene was quantified by Southern blot analyses following digestion with methylation-sensitive enzymes. Estimates of total granule cell number were obtained by counting those cells using unbiased stereological techniques. There was a significant decrease in BDNF mRNA levels in the alcohol-exposed offspring of both ages compared with controls. In addition, the number of olfactory bulb granule cells significantly decreased in the E33 but not the E21 rat pups exposed to alcohol compared with their appropriate aged controls. Finally, BDNF DNA of alcohol-exposed animals was less susceptible to digestion with the methylation-sensitive enzyme HpaII compared with controls, suggesting that the DNA of the alcohol exposed pups was hypermethylated. Our results indicate that exposure to alcohol during early brain development in the rat, a period equivalent to the first two trimesters in humans, can have a detrimental effect on normal development of the olfactory bulb by reducing the number of BDNF-synthesizing neurons. Although the exact mechanism for the alcohol-induced neuronal loss is unknown, the inappropriate transcription of the BDNF gene is one mechanism that may account for the complexity of effects observed in offspring exposed to heavy alcohol exposure in utero.

Quantitative assessment of bacterial contamination of raw milk using bioluminescence.

Enumeration of bacteria in raw milk is of public health and economic importance. Among the proposed rapid methods for assessment of bacterial contamination in raw milk, ATP bioluminescence has proved to be one of the most promising (Griffiths, 1991). Several companies produce ATP bioluminescence reagent kits and equipment for analysing raw milk samples for total bacterial count (Sutherland et al. 1994; Reybroeck & Schram, 1995). The principle of ATP bioluminescent bacterial assay is based on the following assumptions (Olsen, 1991). All living organisms contain ATP, ATP is neither associated with dead cells nor absorbed on to surfaces, colloids and so on, and there is a fairly constant ratio of ATP to biomass/number of cells for all microbial taxa independent of metabolic activity or environmental conditions. Of these assumptions, only the first seems to be indisputable. It is not the number of bacterial cells, but rather the colony forming unit (cfu) that is the denomination used when assessing the microbial quality of milk. For Gram-negative rods of the genus Enterobacteriaceae, a cfu is usually derived from a single cell. However, Gram-positive cocci (staphylococci and streptococci) grow in bunches and chains respectively (Gregg, 1991), and estimation of cell numbers may not give good agreement with the colony counts.Several approaches have been investigated to increase the sensitivity of the bioluminescent method (Pahuski et al. 1991; Sutherland et al. 1994; Reybroeck & Schram, 1995; Froundjian et al. 1999). Although the detection limit achieved by these modifications (104 cfu/ml) may be sufficient for practical use (Bautista et al. 1992; Reybroeck & Schram 1995), the accuracy of the analysis was not significantly improved. The reported values for accuracy of the estimate for cfu/ml in raw milk (Syx) by the bioluminescent method were in the range 0·27–0·87 log units (Bautista et al. 1992; Reybroeck & Schram, 1995). The purpose of the present study was to determine the reasons for the lack of accuracy of the bacterial ATP assay in raw milk.

Improved attachment and spreading in primary cell cultures of the eastern oyster, Crassostrea virginica.

At present, establishment of a cell line from bivalve molluscs has been unsuccessful, and in vitro work is limited to primary cell cultures. We sought to improve attachment and spreading of cells of the eastern oyster, Crassostrea virginica, to aid primary cultures and to assist development of a bivalve cell line. Our objectives were to examine the effects of substrate on ventricle cell viability, attachment, and spreading by testing of collagen I, collagen IV, fibronectin, laminin, poly-D-lysine, and two types of uncoated tissue culture plates (Falcon and Corning). Experiments were conducted by incubating cells with the various substrates for 24 h and 5 d. An assay with a tetrazolium compound (MTS) was used to estimate cell numbers based on metabolic activity. Although differences in MTS assay values for substrate effect on cell viability were detected at 24 h and at 5 d (P > 0.0001), these were attributed to variations in metabolic activity due to different levels of attachment and spreading among treatments. Differences among treatments were detected in attachment and spreading at 24 h and 5 d (for all, P > 0.0001). At 24 h, poly-D-lysine induced the highest levels of attachment and spreading; no other factor performed better than the uncoated Falcon substrate, and collagen I performed most poorly. At 5 d, poly-D-lysine and the uncoated Corning substrate induced significantly higher levels of attachment and spreading than did the uncoated Falcons substrate, and collagen I performed most poorly. From these results, poly-D-lysine best promoted cell attachment and spreading. Fibronectin (at 24 h) and laminin (at 5 d) warrant further study. Along with improvements in medium composition, future work should involve screening of other attachment factors and combinations of factors, including those of bivalve origin.

Modeling the low-LET dose-response of BCR–ABL formation: predicting stem cell numbers from A-bomb data

Formation of the BCR–ABL chromosomal translocation t(9;22)( q34; q11) is essential to the genesis of chronic myeloid leukemia (CML). An interest in the dose-response of radiation induced CML therefore leads naturally to an interest in the dose-response of BCR–ABL formation. To predict the BCR–ABL dose-response to low-linear energy transfer (LET) ionizing radiation, three models valid over three different dose ranges are examined: the first for doses greater than 80 Gy, the second for doses less than 5 Gy and the third for doses greater than 2 Gy. The first of the models, due to Holley and Chatterjee, ignores the accidental binary eurejoining of DNA double-strand break (DSB) free ends (`eurejoining' refers to the accidental restitution of DSB free ends with their own proper mates). As a result, the model is valid only in the limit of high doses. The second model is derived directly from cytogenetic data. This model has the attractive feature that it implicitly accounts for single-track effects at low doses. The third model, based on the Sax–Markov binary eurejoining/misrejoining (SMBE) algorithm, does not account for single-track effects and is therefore limited to moderate doses greater than approximately 2 Gy. Comparing the second model to lifetime excess CML risks expected after 1 Gy, estimates of the number of hematopoietic stem cells capable of causing CML were obtained for male and female atomic bomb survivors in Hiroshima and Nagasaki. The stem cell number estimates lie in the range of 5×10 7–3×10 8 cells.

Monitoring of intracellular ribonucleotide pools is a powerful tool in the development and characterization of mammalian cell culture processes

Efficient cell culture process development for the industrial production of recombinant therapeutics is characterized by constraints which pertain to issues such as costs, competitiveness and the meeting of project timelines. These constraints require tools which can help the developer learn as much as possible as quickly as possible about the cell at hand and identify features of a particular culture which are amenable to improvement. Current on- and off-line monitoring parameters, however useful, provide only late indications (cell concentration, viability) and circumstantial evidence (lactate, ammonia, etc.) with regard to the physiologic status of cells at the time of sampling. The relative intracellular content of purine to pyrimidine nucleotide triphosphates as well as the ratio of UTP to UDP-N-acetylhexosamines have been previously described as sensitive indicators of a cell's metabolic status, growth potential, and overall physiological condition. The sensitivity of such nucleotide ratios and their usefulness in commercially relevant process development and characterization were tested at Boehringer Ingelheim Pharma KG in a large number of fermentations (>80) with a variety of culture modes, cells, and products in scales up to 10,000 litres. Monitoring of these intracellular parameters allows a timely and reliable assessment of cell state and growth potential, which is possible neither by classical cell number and viability measurements nor by a variety of fermentation data typically monitored. The view inside the cell afforded by nucleotide monitoring enables prediction of the behavior of a culture up to 2 days before any hint of physiological changes is given by cell number and viability estimation. In this paper, data relating the growth behavior of CHO and hybridoma cell lines to their nucleotide pools are shown. Two very different processes for the production of recombinant tPA in 10,000-litre bioreactors are compared and characterized with respect to their nucleotide profiles. Examples from industrial process development cases in which intracellular nucleotide information is used to advantage are also presented and discussed. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 357–367, 1999.

Monitoring of intracellular ribonucleotide pools is a powerful tool in the development and characterization of mammalian cell culture processes.

Efficient cell culture process development for the industrial production of recombinant therapeutics is characterized by constraints which pertain to issues such as costs, competitiveness and the meeting of project timelines. These constraints require tools which can help the developer learn as much as possible as quickly as possible about the cell at hand and identify features of a particular culture which are amenable to improvement. Current on- and off-line monitoring parameters, however useful, provide only late indications (cell concentration, viability) and circumstantial evidence (lactate, ammonia, etc.) with regard to the physiologic status of cells at the time of sampling. The relative intracellular content of purine to pyrimidine nucleotide triphosphates as well as the ratio of UTP to UDP-N-acetylhexosamines have been previously described as sensitive indicators of a cell's metabolic status, growth potential, and overall physiological condition. The sensitivity of such nucleotide ratios and their usefulness in commercially relevant process development and characterization were tested at Boehringer Ingelheim Pharma KG in a large number of fermentations (>80) with a variety of culture modes, cells, and products in scales up to 10,000 litres. Monitoring of these intracellular parameters allows a timely and reliable assessment of cell state and growth potential, which is possible neither by classical cell number and viability measurements nor by a variety of fermentation data typically monitored. The view inside the cell afforded by nucleotide monitoring enables prediction of the behavior of a culture up to 2 days before any hint of physiological changes is given by cell number and viability estimation. In this paper, data relating the growth behavior of CHO and hybridoma cell lines to their nucleotide pools are shown. Two very different processes for the production of recombinant tPA in 10,000-litre bioreactors are compared and characterized with respect to their nucleotide profiles. Examples from industrial process development cases in which intracellular nucleotide information is used to advantage are also presented and discussed.

IL-2 and IL-4 counteract budesonide inhibition of GM-CSF and IL-10, but not of IL-8, IL-12 or TNF-alpha production by human mononuclear blood cells.

1. The combination of interleukin-2 (IL-2) and IL-4 reduces the inhibitory effects of glucocorticoids on granulocyte-macrophage colony-stimulating factor (GM-CSF) production, in agreement with the hypothesis that this combination causes glucocorticoid resistance. Whether a general cytokine resistance to glucocorticoids is induced by IL-2 and IL-4 has not been reported. 2. Mononuclear blood cells from healthy individuals were pre-treated with IL-2, IL-4, or IL-2+ IL-4 (31.3-500 U ml(-1)) for 48 h, prior to lipopolysaccharide (LPS; 10 ng ml(-1); 20 h) and budesonide addition. Cytokine levels in the supernatants were analysed using specific immunoassays. DNA content was analysed to estimate cell numbers. 3. GM-CSF production was totally inhibited by budesonide at 10(-8) M in vehicle treated cultures, while IL-10 was inhibited to 33.4+/-4.3% of control. IL-2, IL-4, or IL-2 + IL-4 reduced the inhibitory effects of budesonide on GM-CSF to similar levels (23.7 6.7, 31.6+/-8.5 and 35.1+/-4.3% of control, respectively). IL-2, IL-4, or IL-2 + IL-4 also reduced the inhibitory effects of budesonide on IL-10 production (46.5+/-6.6, 55.9+/-7.3%, and 68.3+/-9.9% of control, respectively). In contrast, IL-8, IL-12 and TNF-alpha production did not become resistant to budesonide. 4. Thus, glucocorticoid resistance induced by IL-2 and IL-4 is not general at the cytokine production level. While the glucocorticoid sensitivity of GM-CSF and IL-10 production decreased, the sensitivity of IL-8, IL-12 or TNF-alpha production was unchanged. Also, the mixture of IL-2 and IL-4 is not crucial for induction of glucocorticoid resistance of GM-CSF production.

Estimation of bacterial cell numbers in humic acid-rich salt marsh sediments with probes directed to 16S ribosomal DNA

The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membrane-bound nucleic acids by using seven group-specific DNA oligonucleotide probes complementary to 16S rRNA coding regions. These included a general eubacterial probe and probes encompassing most members of the gram-negative, mesophilic sulfate-reducing bacteria (SRB). DNA was extracted from sediment samples, and contaminating materials were removed by a series of steps. Efficiency of DNA extraction was 48% based on the recovery of tritiated plasmid DNA added to samples prior to extraction. Reproducibility of the extraction procedure was demonstrated by hybridizations to replicate samples. Numbers of target cells in samples were estimated by comparing the amount of hybridization to extracted DNA obtained with each probe to that obtained with a standard curve of genomic DNA for reference strains included on the same membrane. In June, numbers of SRB detected with an SRB-specific probe ranged from 6.0 x 10(7) to 2.5 x 10(9) (average, 1.1 x 10(9) +/- 5.2 x 10(8)) cells g of sediment-1. In September, numbers of SRB detected ranged from 5.4 x 10(8) to 7.3 x 10(9) (average, 2.5 x 10(9) +/- 1.5 x 10(9)) cells g of sediment-1. The capability of using rDNA probes to estimate cell numbers by hybridization to DNA extracted from complex matrices permits initiation of detailed studies on community composition and changes in communities based on cell numbers in formerly intractable environments.

Distribution and Composition of Microbial Populations in a Landfill Leachate Contaminated Aquifer (Grindsted, Denmark).

> Abstract To investigate whether landfill leachates affected the microbial biomass and/or community composition of the extant microbiota, 37 samples were collected along a 305-m transect of a shallow landfill-leachate polluted aquifer. The samples were analyzed for total numbers of bacteria by use of the acridine orange direct count method (AODC). Numbers of dominant, specific groups of bacteria and total numbers of protozoa were measured by use of the most probable number method (MPN). Viable biomass estimates were obtained from measures of ATP and ester-linked phospholipid fatty acid (PLFA) concentrations. The estimated numbers of total bacteria by direct counts were relatively constant throughout the aquifer, ranging from a low of 4.8 x 10(6) cells/g dry weight (dw) to a high of 5.3 x 10(7) cells/g dw. Viable biomass estimates based on PLFA concentrations were one to three orders of magnitude lower with the greatest concentrations (up to 4 x 10(5) cells/g dw) occurring at the border of the landfill and in samples collected from thin lenses of clay and silt with sand streaks. Cell number estimates based on ATP concentrations were also found to be lower than the direct count measurements (<2.2 x 10(6) cells/g dw), and with the greatest concentrations close to the landfill. Methanogens (Archaea) and reducers of sulfate, iron, manganese, and nitrate were all observed in the aquifer. Methanogens were found to be restricted to the most polluted and reduced part of the aquifer at a maximum cell number of 5.4 x 10(4) cells/g dw. Populations of sulfate reducers decreased with an increase in horizontal distance from the landfill ranging from a high of 9.0 x 10(3) cells/g dw to a low of 6 cells/g dw. Iron, manganese, and nitrate reducers were detected throughout the leachate plume all at maximum cell numbers of 10(6) cells/g dw. Changes in PLFA profiles indicated that a shift in microbial community composition occurred with increasing horizontal distance from the landfill. The types and patterns of lipid biomarkers suggested that increased proportions of sulfate- and iron-reducing bacteria as well as certain microeukaryotes existed at the border of the landfill. The presence of these lipid biomarkers correlated with the MPN results. There was, however, no significant correlation between the abundances of the specific PLFA biomarkers and quantitative measurements of redox processes. The application of AODC, MPN, PLFA, and ATP analyses in the characterization of the extant microbiota within the Grindsted aquifer revealed that as distance increased from the leachate source, viable biomass decreased and community composition shifted. These results led to the conclusion that the landfill leachate induced an increase in microbial cell numbers by altering the subsurface aquifer so that it was conducive to the growth of methanogens and of iron-and sulfate-reducing bacteria and fungi.

Estimation of PC12 cell numbers with acid phosphatase assay and mitochondrial dehydrogenase assay: dopamine interferes with assay based on tetrazolium.

Both the acid-phosphatase and mitochondrial dehydrogenase assay have been used to quantify cell numbers. The commonly used acid-phosphatase assay uses p-nitrophenyl phosphate as a substrate, while the mitochondrial dehydrogenase assay is based on the conversion of tetrazolium to formazan. Our experimental results showed that the former assay was more sensitive in detecting small numbers of PC12 cells (200-10,000 cells/well), whereas the latter was useful for larger numbers of cells (2000-40,000 cells/well). The number of PC12 cells decreased after dopamine treatment, according to the acid-phosphatase assay and by direct cell counts under a light microscope. However, the optical densities measured by the mitochondrial dehydrogenase assay increased after dopamine treatment. We tried to clarify discrepancies between the two assays, since dopamine is an important neurotransmitter and both assays are commonly used to estimate cell numbers. To elucidate the interference between dopamine and tetrazolium salt, cell-free control experiments were performed. Dopamine and other catecholamines (adrenaline and noradrenaline) reacted with tetrazolium and, thus, produced a false positive reaction in the assay. We therefore conclude that the tetrazolium assay is not a suitable method for evaluating the number of catecholamine-treated cells, while the acid-phosphatase assay is reliable and sensitive.

Antiproliferative Effect of the Tripeptide PyroGlu-Phe-GlyNH2on Murine Melanocytes: Transitory Delay of Cell Growthin Vitroand the Cell Cycle Specificity

Cell growth and differentiation in melanocyte cell populations are regulated by a wide range of bioactive substances. Recently, the tripeptide pyroGlu-Phe-GlyNH2which inhibits melanocyte growthin vitrowas identified in both murine nontransformed melanocytes and malignant melanoma cells. The present study was undertaken to investigate the cell cycle specificity as well as the growth inhibitory profile of the tripeptide after a single or repeated administration to melanocyte cultures. Dose-related effects of the peptide were studied using three different bioassay systems: estimation of cell number, DNA synthesis, and cell flux into mitosis. Growth of melanocyte cultures as well as melanocyte mitotic activity were found to be reduced significantly by the tripeptide at two separate dose levels (10−11and 10−14–10−15M). Growth inhibition of melanocyte population did not last long: less than 36 h after the first and less than 24 h after the second peptide addition to the cultures. The level of DNA synthesis in melanocytes remained unchanged after a single peptide administration. The findings indicate that the tripeptide pyroGlu-Phe-GlyNH2causes transitory delay of cell growth in cultured melanocyte population resulting from a reversible inhibition of melanocyte transition from the G2-phase of the cell cycle into mitosis.

Protein kinase C is involved in cholecystokinin octapeptide-induced proliferative action in rat glioma C6 cells.

Chotecystoknin octapeptide (CCK-8) has been shown to stimulate DNA synthesis in rat glioma C6 cells by activation of CCKB type receptors. However, the signalling pathways contributing to this proliferative action in C6 cells have not been investigated thus far. This study demonstrated that stimulation of rat glioma C6 cells with CCK-8S resulted in activation of protein kinase C isozymes betaI, betaII, gamma and zeta. The participation of protein kinase C in the CCK-8S-induced effect on C6 cell growth was demonstrated by measurement of [3H]thymidine incorporation and estimation of cell number. The data indicate that CCK-8S stimulates growth in rat glioma C6 cells by a protein kinase C-dependent mechanism.

A very rapid enzyme immunoassay for confirmation of rhizobial identity and estimation of cell numbers in fresh broth culture

An enzyme immunoassay has been developed that confirms identity and estimates cell numbers of Bradyrhizobium japonicum and Rhizobium leguminosarum bv. viciae in broth culture. Total testing time required is less than 10 min and no specialized equipment is required. The assay involves a simultaneous and brief reaction of cells, primary antibody, and secondary antibody - enzyme conjugate in aqueous suspension, after which the cells are captured on a 0.45- µm-pore-size syringe filter membrane. Upon addition of enzyme substrate, cell-bound antibody-enzyme conjugate converts the substrate to a blue-green reaction product within the transparent body of the syringe filter. The assay can be quantified by stopping the reaction with acid, extracting the coloured substrate reaction product from the syringe filter, and measuring absorbance using a spectrophotometer. The assay was developed using cultures of rhizobia and bradyrhizobia but should be applicable to liquid cultures of most microorganisms.Key words: Bradyrhizobium, enzyme immunoassay, Rhizobium, syringe filter.

A very rapid enzyme immunoassay for confirmation of rhizobial identity and estimation of cell numbers in fresh broth culture

An enzyme immunoassay has been developed that confirms identity and estimates cell numbers of Bradyrhizobium japonicum and Rhizobium leguminosarum bv. viciae in broth culture. Total testing time required is less than 10 min and no specialized equipment is required. The assay involves a simultaneous and brief reaction of cells, primary antibody, and secondary antibody - enzyme conjugate in aqueous suspension, after which the cells are captured on a 0.45- µm-pore-size syringe filter membrane. Upon addition of enzyme substrate, cell-bound antibody-enzyme conjugate converts the substrate to a blue-green reaction product within the transparent body of the syringe filter. The assay can be quantified by stopping the reaction with acid, extracting the coloured substrate reaction product from the syringe filter, and measuring absorbance using a spectrophotometer. The assay was developed using cultures of rhizobia and bradyrhizobia but should be applicable to liquid cultures of most microorganisms.Key words: Bradyrhizobium, enzyme immunoassay, Rhizobium, syringe filter.

A Most Probable Number method (MPN) for the estimation of cell numbers of heterotrophic nitrifying bacteria in soil

A Most Probable Number (MPN) method was developed allowing for the first time estimation of populations of bacteria capable of heterotrophic nitrification. The method was applied to an acidic soil of a coniferous forest exhibiting nitrate production. In this soil nitrate production was unlikely to be catalyzed by autotrophic nitrifiers, since autotrophic ammonia oxidizers never could be detected, and autotrophic nitrite oxidizers were usually not found in appreciable cell numbers. The developed MPN method is based on the demonstration of the presence/absence of nitrite/nitrate produced by heterotrophic nitrifying bacteria during growth in a complex medium (peptone-meat-extract softagar medium) containing low concentrations of agar (0.1%). Both the supply of the growing cultures in MPN test tubes with sufficient oxygen and the presence of low agar concentrations in the medium were found to be favourable for sustainable nitrite/nitrate production. The results demonstrate that in the acidic forest soil the microbial population capable of heterotrophic nitrifcation represents a significant part of the total aerobic heterotrophic population. By applying the developed MPN method, several bacterial strains of different genera not previously described to perform heterotrophic nitrification have been isolated from the soil and have been identified by bacterio-diagnostic tests.

Effects of unilateral olfactory deprivation in the developing opossum, Monodelphis domestica.

Unilateral naris closure in young rodents leads to striking alterations in the development of the ipsilateral olfactory system. One of the most pronounced effects is a 25% reduction in the size of the experimental olfactory bulb, a change that stems in part from decreased cell survival. Since naris occlusion in rodents alters the system more during development than in adulthood, we investigated the consequences of olfactory deprivation in a species that is born in a very immature state, Monodelphis domestica. In this pouchless marsupial, offspring are born after a short 14-day gestation. In the present study, the thymidine analogue bromodeoxyuridine was used to examine early postnatal neurogenesis in the olfactory bulb. Unlike rats and mice, neurogenesis of the main output neurons (the mitral cells) continues into postnatal life. Unilateral naris closure was begun on postnatal day 4 (P4) or P5 in Monodelphis and continued for 30 or 60 days. Laminar volume measurements revealed a significant reduction in the size of the experimental bulb following 60, but not 30, days of early olfactory deprivation. Mitral cell number estimates indicated a significant reduction after both 30 and 60 days of naris closure. The immaturity of Monodelphis offspring may render the population of mitral cells susceptible to the effects of olfactory deprivation. These findings suggest that afferent activity plays a role in the survival of all bulb neurons, irrespective of cell class.

Assessment of the microbial potential for nitrate-enhanced bioremediation of a JP-4 fuel-contaminated aquifer

A site that was contaminated with JP-4 jet fuel was characterized microbiologically to assess the feasibility of nitrate-enhanced bioremediation. The results of microcosm studies indicated that the mean pseudo zero-order rate constants for alkylbenzene biodegradation and NO3−-N removal were 1.2 and 2.4 mg L−1 per day, respectively. Several alkylbenzenes were removed to a greater extent in samples contaminated with residual JP-4 than in unexposed samples and samples downgradient of the spill; benzene was recalcitrant in all samples. Numbers of total heterotrophs, JP-4-degraders, oligotrophs, total denitrifiers, denitrifiers growing in the presence of JP-4, estimates of cell number by analysis of phospholipid fatty acids, direct counts and aerobic and anaerobic protozoa were determined; however, numbers of microorganisms were not reliable predictors of alkylbenzene biodegradation activity. The presence of aerobic and anaerobic protozoa suggests that protozoa may be active under a variety of different electron acceptor conditions. The results of the characterization study indicated that the site was amenable to nitrate-enhanced bioremediation.

Lipopolysaccharide が骨芽細胞の増殖と分化におよぼす影響

Lipopolysaccharide (LPS) is one of the candidates for the alveolar bone loss accompanied with periodontal disease. The purpose of this study is to assess the effect of LPS on cell proliferation and the differentiation of osteoblastic cells in cell culture. Osteoblastic cells were prepared from rat calvaria, and the cells were cultured with LPS in the presence and the absence of ascorbic acid. After the culture, the cell proliferation was examied by scoring colony formed or estimating cell number using cell counting kit. The osteoblastic differentiation was examined by measuring ALP activity of cell lysate. The results obtained were as follows : 1. In osteoblastic cell culture at low cell density, many colonies were formed. More than half of these colonies were ALP positive from the histochemical stainning. Mineralized nodules were appeared in the culture adding ascorbic acid and β-glycerophophate. 2. LPS (0.1, 1, 10μg/ml) increased cell number of osteoblastic cells in colony assay and in the assay cell counting kit. 3. LPS greatly affected ALP activity of osteoblastic cells. The activity was also positively and negatively influenced by the addition of ascorbic acid and cell density. Thus, LPS increased osteoblastic cell proliferation and affected ALP activity of osteoblastic cells. These results suggest that LPS modulate alveolar bone loss accompanied with periodontal disease.

Cellular defense mechanisms in the udder and lactation of goats.

Migration of neutrophils into mammary tissue provides the first immunological line of defense against bacteria that penetrate the physical barrier of the teat canal. Evasion of neutrophil defenses provides an opportunity for invading bacteria to become established. Depletion of neutrophils results in a dramatic increase in susceptibility to intramammary infection. Numerous cytoplasmic particles are shed from the apical surface of mammary secretory cells during milk secretion in goats. Only those counting methods that are specific for deoxyribonucleic acid can distinguish cell-like particles from somatic cells and thereby give reliable estimates of somatic cell numbers in goat milk. Unlike in milk from dairy cows, the somatic cell count in goat milk is influenced by the presence of nucleated cytoplasmic particles, stage of lactation, parity, and caprine arthritis-encephalitis. Investigations indicate that a dry period is necessary for optimal milk production in dairy cows but may not be necessary in goats. However, in many other respects regulation of bovine and caprine lactation seems to be quite similar. Studies have demonstrated additive galactopoietic effects of growth hormone and frequent milking in both species and a recently isolated chemical feedback inhibitor of lactation seems effective across both species. Increasing lactational performance has the potential for decreasing milk somatic cell counts in late lactation.

Postnatal development of a sexually dimorphic, hypothalamic nucleus in gerbils: a stereological study of neuronal number and apoptosis.

Steroid-sensitive, vocal courtship behavior is a function of a specific, hypothalamic nucleus, the sexually dimorphic area pars compacta (SDApc) in the male adult gerbil. Gender-related differences in the number of neurons in this nucleus are evident immediately after birth. By using unbiased stereological estimates of cell numbers in Nissl-stained, paraffin-wax sections of brain, we investigated the mechanisms differentiating cell number between the sexes in the SDApc on postnatal days 0, 3, 6, and 15. Cell death, identified by pyknosis, was greatest in the SDApc between days 0-3 in males, whereas in females, maximum values were reached between days 3-6. Similarly, the ratio of pyknotic to normal neurons peaked between days 0-3 in males and 3-6 in females but then declined in both sexes. Pyknotic cells were seldom seen in either sex by day 15. Morphological characteristics of apoptosis including chromatin condensation, cell fragmentation, and ingestion of apoptic bodies by macrophages were all demonstrated by transmission electron microscopy. Macrophages showed specific morphological characteristics of microglia. Cell division (mitosis) was identified in the SDApc during postnatal days 0, 3, and 6 but the numbers of mitotic figures were low, negligible on day 15, and similar between the sexes. These results demonstrate that cell death and proliferation occur simultaneously in the neonatal gerbil brain. The stereological estimates of cell death in the developing SDApc indicated a lower incidence of neuronal death occurring earlier in males than in females.