Effect of dispersing oil phase on the biodegradability of a solid alkane dissolved in non-biodegradable oil

Acinetobacter sp. CR was grown on a model oil, which consisted of an inert oil matrix of pristane with n-heneicosane dissolved in it as the sole carbon source, in a stirred-tank bioreactor. This bacterium takes up substrates from the oil phase by direct contact with the oil phase. A previously established mathematical model was applied to reveal the effect of agitation conditions on the growth and n-alkane degradation kinetics of the bacterium. Higher impeller speed resulted in both lower microbial growth and lower n-alkane degradation rate of the bacterium, although it increased the specific surface area of the oil, which was measured by a previously developed device. This result was due to the decreased number of cells adhering to the oil surface, i.e., intense agitation inhibited the adhesion of cells to the oil surface. The addition of a surfactant below a critical micelle concentration (CMC) inhibited the degradation of n-heneicosane dissolved in pristane, although the biodegradability of the substrate recovered gradually with the increase in the dose of surfactant over CMC. The results suggest that efforts to increase the specific surface area of the oil phase have the undesirable result of inhibiting oil degradation when the dominant microbial degraders take up substrates in oil by direct contact with the oil. Electronic Publication     

The biology and cytogenetics of multiple myeloma

Despite the advances in our knowledge of myeloma cell biology, our understanding of myeloma pathogenesis is still incomplete. In this review, we present a summary of the cellular and molecular aspects of B-cell development and immunoglobulin (lg) gene rearrangement which have been important in defining the characteristics of the myeloma plasma cell (MPC). The PMC has undergone variable gene recombination, somatic hypermutation and isotype switching, and is therefore at a postgerminal center stage of development. The finding of preswitch clonal cells and isotype variants have raised interesting questions about the cell of origin of myeloma, for which no conclusive data is as yet available. However much information has been obtained about the chromosomal and genetic aberrations in myeloma, including monosomy 13, Ig heavy chain (IgH) switch region translocations, numerical abnormalities and a multitude of heterogeneous changes. A variety of techniques have been developed to overcome the insensitivity of conventional karyotyping, utilizing molecular cytogenetic strategies ranging from the delineation of precise loci by fluorescent in situ hybridization, a more "global" assessment of the genome by multicolor spectral karyotyping, to the quantitation of chromosomal material of specific origin by comparative genomic hybridization. Whether the abnormalities detected represent oncogenic insults, are involved in disease progression or are simply "by-products" of genetic instability is still unclear. For IgH translocations, the role of candidate genes such as Cyclin D1 and FGFR3 has been studied extensively by quantitating their expression and assessment of their oncogenicity (e.g. for FGFR3) in animal models. The significance of other aberrations such as c-myc, ras and p53 has also been investigated. With the advent of oligonucleotide microarrays, the expression of thousands of genes can be efficiently examined. So far, this approach seems promising in defining subgroups of different disease behavior, and may highlight specific genes and molecular mechanisms which are important in myeloma pathogenesis.     

A cyclic AMP-dependent pathway regulates the expression of acetylcholinesterase during myogenic differentiation of C2C12 cells

The expression of acetylcholinesterase (AChE) is markedly increased during myogenic differentiation of C2C12 myoblasts to myotubes; the expression is mediated by intrinsic factor(s) during muscle differentiation. In order to analyze the molecular mechanisms regulating AChE expression during myogenic differentiation, a approximately 2.2-kb human AChE promoter tagged with a luciferase reporter gene, namely pAChE-Luc, was stably transfected into C2C12 cells. The profile of promoter-driven luciferase activity during myogenic differentiation of C2C12 myotubes was found to be similar to that of endogenous expression of AChE catalytic subunit. The increase of AChE expression was reciprocally regulated by a cAMP-dependent signaling pathway. The level of intracellular cAMP, the activity of cAMP-dependent protein kinase, the phosphorylation of cAMP-responsive element binding protein and the activity of cAMP- responsive element (CRE) were down-regulated during the myotube formation. Mutating the CRE site of human AChE promoter altered the original myogenic profile of the promoter activity and its suppressive response to cAMP. In addition, the suppressive effect of the CRE site is dependent on its location on the promoter. Therefore, our results suggest that a cAMP-dependent signaling pathway serves as a suppressive element in regulating the expression of AChE during early myogenesis.     

Kinetic characterization of yeast pyruvate carboxylase isozyme pyc1

Yeast (Saccharomyces cerevisiae) is unusual in being the only organism thus far identified as having two genes for pyruvate carboxylase. The expression of the two isozymes Pyc1 and Pyc2 appears to be differentially regulated, and since both are expressed cytoplasmically, this suggests that they have different properties. To the present, little has been done to characterize these isozymes, and almost all of the published kinetic information on yeast pyruvate carboxylase comes from measurements of enzyme prepared from bakers' yeast which is likely to be a mixture of both isozymes. Here we have measured basic kinetic parameters for Pyc1 and found that the K(a) of this isozyme for acetyl CoA is in the order of 8-10-fold higher than previously recorded, suggesting that Pyc1 and Pyc2 may be differentially regulated by this effector. Pyc1 is highly dependent on the presence of acetyl CoA for activity and in this respect is similar to chicken liver pyruvate carboxylase. However, unlike the chicken liver enzyme, the quaternary structure of the enzyme is quite stable in the absence of acetyl CoA, and the major locus of action of this effector appears to lie outside of the stimulation of the biotin carboxylation reaction.     

Signaling mechanisms of the D3 dopamine receptor

A substantial body of evidence shows the capacity of the dopamine D3 receptor to couple functionally to G proteins when expressed in an appropriate milieu in heterologous expression systems. In these systems, activation of D3 receptors inhibits adenylate cyclase, modulates ion flow through potassium and calcium channels, and activates kinases, most notably mitogen-activated protein kinase. Coupling to Gi/Go is implicated in many of these effects, but other G proteins may contribute. Studies with chimeric receptors implicate the third intracellular loop in the mediation of agonist-induced signal transduction. Finally, D3-preferring drugs modulate expression of c-fos in neuronal cultures and brain. Signaling mechanisms of the D3 receptor in brain, however, remain to be definitively determined.     

Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors

The authors have constructed an array of 12 piezoelectric ejectors for printing biological materials. A single-ejector footprint is 8 mm in diameter, standing 4 mm high with 2 reservoirs totaling 76 micro L. These ejectors have been tested by dispensing various fluids in several environmental conditions. Reliable drop ejection can be expected in both humidity-controlled and ambient environments over extended periods of time and in hot and cold room temperatures. In a prototype system, 12 ejectors are arranged in a rack, together with an X - Y stage, to allow printing any pattern desired. Printed arrays of features are created with a biological solution containing bovine serum albumin conjugated oligonucleotides, dye, and salty buffer. This ejector system is designed for the ultra-high-throughput generation of arrays on a variety of surfaces. These single or racked ejectors could be used as long-term storage vessels for materials such as small molecules, nucleic acids, proteins, or cell libraries, which would allow for efficient preprogrammed selection of individual clones and greatly reduce the chance of cross-contamination and loss due to transfer. A new generation of design ideas includes plastic injection molded ejectors that are inexpensive and disposable and handheld personal pipettes for liquid transfer in the nanoliter regime.     

CK2 protein kinase is stimulated and redistributed by functional herpes simplex virus ICP27 protein

It has been shown previously (S. Wadd, H. Bryant, O. Filhol, J. E. Scott, T.-T. Hsieh, R. D. Everett, and J. B. Clements, J. Biol. Chem. 274:28991-28998, 2000) that ICP27, an essential and multifunctional herpes simplex virus type 1 (HSV-1) protein, interacts with CK2 and with heterogeneous ribonucleoprotein K (hnRNP K). CK2 is a pleiotropic and ubiquitous protein kinase, and the tetrameric holoenzyme consists of two catalytic alpha or alpha' subunits and two regulatory beta subunits. We show here that HSV-1 infection stimulates CK2 activity. CK2 stimulation occurs at early times after infection and correlates with redistribution of the holoenzyme from the nucleus to the cytoplasm. Both CK2 stimulation and redistribution require expression and cytoplasmic accumulation of ICP27. In HSV-1-infected cells, CK2 phosphorylates ICP27 and affects its cytoplasmic accumulation while it also phosphorylates hnRNP K, which is not ordinarily phosphorylated by this kinase, suggesting an alteration of hnRNP K activities. This is the first example of CK2 stimulation by a viral protein in vivo, and we propose that it might facilitate the HSV-1 lytic cycle by, for example, regulating trafficking of ICP27 protein and/or viral RNAs.     

Establishment and maintenance of gammaherpesvirus latency are independent of infective dose and route of infection

Gammaherpesviruses such as Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are important human pathogens that establish long-term latent infections. Understanding of the initiation and maintenance of latent infections has important implications for the prevention and treatment of gammaherpesvirus-related diseases. Although much is known about gammaherpesvirus pathogenesis, it is unclear how the infectious dose of a virus influences its ability to establish latent infection. To examine the relationship between the infectious dose and gammaherpesvirus latency, we inoculated wild-type mice with 0.01 to 10(6) PFU of murine gammaherpesvirus 68 (gammaHV68) and quantitatively measured latency and acute-phase replication. Surprisingly, during latency, the frequencies of ex vivo reactivation were similar over a 10(7)-fold range of doses for i.p. infection and over a 10(4)-fold range of doses for intranasal infection. Further, the frequencies of cells harboring viral genome during latency did not differ substantially over similar dose ranges. Although the kinetics of acute-phase replication were delayed at small doses of virus, the peak titer did not differ significantly between mice infected with a large dose of virus and those infected with a small dose of virus. The results presented here indicate that any initiation of infection leads to substantial acute-phase replication and subsequent establishment of a maximal level of latency. Thus, infections with doses as small as 0.1 PFU of gammaHV68 result in stable levels of acute-phase replication and latent infection. These results demonstrate that the equilibrium level of establishment of gammaherpesvirus latency is independent of the infectious dose and route of infection.     

Identification and characterization of novel rat and human gonad-specific organic anion transporters

We have isolated three novel organic anion transporter cDNAs designated rat GST-1 (gonad-specific transporter), rat GST-2, and human GST, expressed at high levels in the testis. Rat GST-1, GST-2, and human GST consist of 748, 702, and 719 amino acids, respectively, and all molecules possess the 12 predicted transmembrane domains, which is a common structure of organic anion transporters. Northern blot analyses and in situ hybridization revealed that both of the rat molecules are highly expressed in the testis, especially in Sertoli cells, spermatogonia, and Leydig cells. Weak signals are also detected in the epididymis and ovary in adult rat. The exclusive expression of human GST mRNA in the testis was confirmed by RT-PCR. The pharmacological experiments of Xenopus laevis oocytes injected with the respective rat GST-1- and GST-2-cRNAs revealed that both rat GST-1 and GST-2 transport taurocholic acid, dehydroepiandrosterone sulfate, and T4 with Michaelis-Menten kinetics (taurocholic acid, Km = 8.9 and 2.5 microm, dehydroepiandrosterone sulfate, Km = 25.5 and 21.microm, and T4, Km = 6.4 and 5.8 for rat GST-1 and GST-2, respectively). T3 was also transported by rat GST-1 and GST-2. These data suggest that rat GST-1 and GST-2 might be one of the molecular entities responsible for transporting dehydroepiandrosterone sulfate and thyroid hormones involved in the regulation of sex steroid transportation and spermatogenesis in the gonad.