Autoradiographic studies of nicotinic acid utilization in human-mouse heterokaryons and inhibition of utilization in newly-formed hybrid cells

Although most mammalian cell lines can utilize either nicotinic acid or nicotinamide for the biosynthesis of nicotinamide adenine dinucleotide (NAD), thymidine kinase-deficient, mouse 3T3–4F cells are unable to utilize nicotinic acid. When 3T3–4E cells were fused with human D98/AH2 cells, autoradiography showed that the resultant heterokaryons synthesized NAD from nicotinic acid at rates comparable to the human parental cell. The rate of nicotinic acid utilization in heterokaryons remained unchanged over the fourday period of study following cell fusion. In contrast to the results observed with heterokaryons, nicotinic acid utilization was markedly reduced in hybrid cells. Of 100 hybrid clones examined at four or five days following cell fusion, 60 utilized nicotinic acid at rates less than one tenth that of the parental human cell. Similar results were observed in hybrid clones at nine or ten days following fusion. Uniformly high rates of NAD biosynthesis were observed in hybrid clones with nicotinamide as the precursor. This excludes the possibility that the reduction in nicotinic acid utilization in hybrid cells is due to a general metabolic dysfunction. The biochemical mechanism by which nicotinic acid utilization is markedly reduced has not been determined with certainty, however, several observations suggest genetic suppression.     

Kinetics of mucopolysaccharide and glycoprotein synthesis by chick embryo chondrocytes. Effect of D-glucose concentration in the culture medium.

Late log cultures of chick embryo vertebral chondrocytes in Dulbecco's Modified Eagle's Medium with 10% fetal calf serum consume D-glucose from the culture medium at a rate of approximately 0.40 mumol per h per 10(6) cells. When the D-glucose concentration in the medium drops below 1 mumol per ml the glycogen stores are rapidly exhausted, and cell growth ceases. 35SO4(2)- is incorporated into chondroitin-6-SO4 and chondroitin-4-SO4 at linear rates of 1.2 and 0.4 nmol per h per 10(6) cells, respectively, until the D-glucose level in the medium drops below 1 mumol per ml, but there is always a slight lag in the initial appearance of chondroitin-4-SO4. Throughout the period of 35SO4 2- labeling, the amount of chondroitin-6-SO4 that is recovered in the cells exceeds the amount that is recovered in the medium, but the opposite is true for chondroitin-4-SO4. However, when cells prelabeled with 35SO4(2-) are then transferred to a label-free medium, the secretion of chondroitin sulfates proceeds at much slower rates, and the kinetics of chondroitin-6-SO4 and chondroitin-4-SO4 secretion are very similar. In this chase experiment the chondroitin sulfates are recovered quantitatively after a 24-h incubation period, indicating that these embryonic chondrocytes do not degrade the chondroitin sulfates under these culture conditions. The rate of incorporation of counts from D-[14C]glucosamine into mucopolysaccharides and glycoproteins increase with time. This nonlinear rate results from a progressive increase in the specific activity of the UDP-N-acetyl-D-[14C]hexosamine pool as the D-glucose in the culture medium is depleted. A linear relationship is demonstrated between the logarithm of the 14C counts per min per nmol of UDP-N-acetylhexosamine and the logarithm of the concentration of D-glucose in the culture medium over a range of 1 to 20 mumol of D-glucose per ml. The relative rates of appearance of counts from 35SO4(2-) and D-[14]glucosamine in chondroitin 4-SO4 and chondroitin-6-SO4 are used to calculate the specific activity of the UDP-N-acetyl-D-[14C]hexosamine pool at each stage in the labeling period. The resulting values are then used to calculate the rates of synthesis of the nonsulfated polymers, namely, chondroitin, hyaluronic acid, and glycoprotein.     

C2 deficiency in man. genetic relationship to a mixed lymphocyte reaction determinant (7a*)

Three unrelated individuals with, respectively, lupus erythematosus, polyarteritis, and membranoproliferative glomerulonephritis and totally deficient in the second component of complement are demonstrated to be mutually poorly reactive in mixed lymphocyte culture and homozygous for the mixed lymphocyte reaction determinant (MLR-S or LD) short 7a (7a^*). The gene controlling the elaboration of C2 in man is shown to be separate from, and probably to map outside of, the second locus ofHL-A and theMLR-S locus. Genetic linkage disequilibrium is strongly suggested between HL-A 10, W18, 7a^*, and C2 deficiency.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

Parrotfish sex ratios recover rapidly in Bermuda following a fishing ban

Parrotfishes are an ecologically and commercially important teleost group whose grazing contributes to maintaining coral-dominated states on hermatypic reefs. However, overfishing has skewed sex ratios of Atlantic parrotfishes because fishing has disproportionate impacts on larger individuals, and males are generally larger than females. Whether protection from fishing may allow sex ratios to return to equilibrium is unknown, as fishing can induce irreversible ecological and/or evolutionary shifts. Bermuda banned trap fishing in 1990, creating a unique opportunity to analyse long-term responses of Atlantic parrotfishes to release from fishing. We found that sex ratios of four common parrotfishes were initially skewed, with male proportions ranging from 0.04 to 0.18. However, male proportions rebounded within 3–4 yr, equilibrating at values ranging from 0.36 to 0.54, similar to those reported at unfished sites in the region. Our results are encouraging for regional efforts to recover lost grazing function by restoring overfished herbivore populations.     

Comparative proteome analysis using amine-reactive isobaric tagging reagents coupled with 2D LC/MS/MS in 3T3-L1 adipocytes following hypoxia or normoxia

Hypoxia during the expansion of adipocytes is known to contribute both to the secretion of multiple inflammation-related adipokines as well as to obesity. We therefore investigated the nature of protein changes occurring in adipocytes during hypoxia by observation of the intracellular proteins that are expressed in 3T3-L1 adipocytes. Lysates were utilized for quantitative proteome analysis using isobaric tags for relative and absolute quantitation (iTRAQ) combined with peptide separation by multi-dimensional liquid chromatography. Antioxidants and elongation factors, as well as glycolytic enzymes were increased in hypoxic adipocytes. These changes were supported by similar changes suggested by real-time PCR. The proteins showing changes are all potential targets for revering the mechanism behind the phenomenon of induction of obese adipocytes by hypoxia. This study can therefore aid in defining the proteomic changes that occur in adipocytes in response to oxygen stress, and can further characterize adipocyte metabolism and adaptation to low oxygen conditions.     

Long-term use of high-efficiency vacuum cleaners and residential airborne fungal-spore exposure

Exposure to fungal allergens is an important contributor to allergic respiratory disease, but information on the efficacy of residential fungal allergen-avoidance in allergic-disease management is lacking. Using vacuum cleaners with high-efficiency exhaust filtration is one method recommended for reducing residential allergen exposure levels, but their use to reduce fungal-spore exposure levels has not been evaluated. To evaluate the effectiveness of high-efficiency vacuuming to control airborne fungal-spore levels, fungal bioaerosols were repeatedly assessed over the course of 10 months in homes randomly assigned to groups using either conventionally filtered (control) or high-efficiency-filtered vacuum cleaners for routine vacuum cleaning. Air samples were analyzed for three fungal-spore categories representing taxa with predominantly outdoor sources and one representing taxa that commonly have indoor sources. In a two-way analysis of variance, sampling period had a significant effect on mean levels of all fungal-spore categories. Vacuum cleaner type had a marginally significant effect on the indoor spore category, with one high-efficiency vacuum group mean (of three) significantly lower than one control mean. No effect was observed of vacuum cleaner type on outdoor spore categories. Including home-environment variables in analysis of covariance models strengthened the effect of the vacuum-type treatment on the indoor spore category, with no effect on the three outdoor spore categories. Decreased indoor spore levels vs. controls were only observed in high-efficiency vacuum groups during the last sampling period, at the end of the heating season. The results suggest that using a vacuum with high-efficiency filtered exhaust could have some modest effectiveness in controlling airborne fungal-spore exposure in homes when infiltration of outdoor air is very limited.