Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.

Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K(m) values for the two substrates UDP-GlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 x 10(5) to 1 x 10(6) Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 x 10(6) Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions.

The gene structure and promoter sequence of mouse hyaluronan synthase 1.

The structure and organization of mouse hyaluronan synthase 1 gene, HAS1 were determined by direct sequencing of lambda phage clones carrying the entire gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon-intron boundaries and all of the exons. This gene spans about 11kb of genomic DNA and consists of 5 exons and 4 introns. A similarity in the exon-intron organization was found between the genes of mouse HAS1 and Xenopus laevis DG42 which was recently identified as Xenopus hyaluronan synthase. The transcription initiation site was determined by rapid amplification of the cDNA ends (5'-RACE). Position +1 is located 55 nucleotides upstream of the ATG initiation codon. The promoter region of the HAS1 gene has no typical TATA box, but contains a CCAAT box located 190 nucleotides upstream of the transcription initiation site. Further analysis of 1.4 kb of the 5' flanking region revealed several potential binding motifs for transcription factors. This information about the gene structure may be useful for further studies on the promoter activity.

Oscillations of two competing microbial populations in configurations of two interconnected chemostats.

It is known that, when two microbial populations competing for a single rate-limiting nutrient are grown in a spatially uniform environment, such as a single chemostat, with competition being the only interaction between them, they cannot coexist, but eventually one of the two populations prevails and the other becomes extinct. Spatial heterogeneity has been suggested as a means of obtaining coexistence of the two populations. A configuration of two interconnected chemostats is a simple model of a spatially heterogeneous environment. It has been shown that, when Monod's model is used for the specific growth rates of the two populations, steady-state coexistence can be obtained in such systems for wide ranges of operating conditions. In the present work, we study a model of microbial competition in configurations of interconnected chemostats and we show that, if a substrate inhibition model is used for the specific growth rates of the two populations, coexistence in a periodic state is also possible. The analysis of the model is done by numerical bifurcation theory methods.

Adaptive fuzzy control of nutrients concentration in fed-batch culture of mammalian cells.

An adaptive fuzzy controller was developed to control the glucose and glutamine concentrations in the reactor constant at the desired level. The parameter values of the controller change during the cultivation according to the culture phase which was detected by the lactate concentration. Cultivations with different glucose and glutamine set point concentrations of a recombinant BHK anchorage-dependent cell line were performed in a fed-batch reactor on-line connected with an HPLC system. Glucose and glutamine concentrations were satisfactorily controlled at each set point during all cultivation periods. Ammonia had a determining effect on productivity since it inhibited cell growth and protein specific production. Ammonia production increased with an increase of glutamine or a decrease of glucose set point concentrations, indicating the importance of glucose to glutamine ratio for the optimization of productivity in mammalian cell cultures.

Coexistence of three competing microbial populations in a chemostat with periodically varying dilution rate.

Coexistence of three microbial populations engaged in pure and simple competition is not possible in a chemostat with time-invariant operating conditions under any circumstances. It is shown that by periodic variation of the chemostat dilution rate it is possible to obtain a stable coexistence state of all three populations in the chemostat. This is accomplished by performing a numerical bifurcation analysis of a mathematical model of the system and by determining its dynamic behavior with respect to its operating parameters. The coexistence state obtained in the periodically operated chemostat is usually periodic, but cases of quasi-periodic and chaotic behavior are also observed.

Periodic, quasi-periodic, and chaotic coexistence of two competing microbial populations in a periodically operated chemostat.

It is well known that when two microbial populations competing for a single rate-limiting nutrient are grown in a chemostat with time-invariant inputs, with competition being the only interaction between them, they cannot coexist, but eventually one of the two populations prevails and the other becomes extinct. It has been suggested that periodic variation of one of the chemostat's operating parameters can stabilize the coexistence state of the two microbial populations. A systematic numerical study of the model equations describing microbial competition in a chemostat with periodically varying dilution rate is performed, and it is shown that coexistence of the competing microbial populations is obtained for a wide range of operating conditions. The coexistence state is usually in the form of limit cycle oscillations. However, cases of chaotic behavior resulting from successive period doublings and quasi-periodicity are also observed.

Cisplatin stimulates the expression from the human immunodeficiency virus long terminal repeat sequences in human fibroblasts.

A recombinant plasmid carrying the long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1) linked to the reporter chloramphenicol acetyl transferase (CAT) gene was stably introduced into human fibroblasts. The transfectant cells expressed CAT activity from the HIV LTR. The response to anti-neoplastic drugs, i.e. cisplatin, a platin derivative, and hexadecylphosphocholine, was studied. It was found that at 5 x 10(-6) M concentrations cisplatin stimulates by 2.2-fold the expression of CAT from the HIV LTR. Our results extend our observations on the effect of cisplatin on HIV LTR in rodent fibroblast cells and suggest caution against therapy including cisplatin in the treatment of AIDS patients.