Codominant translational mutants of Chinese hamster ovary cells selected with diphtheria toxin.

Diphtheria toxin-resistance markers in two translational mutants, CH-RE1.22c, possessing no toxin-sensitive EF-2 (class IIa), and CH-RE1.32, with 50% toxin-sensitive and 50% toxin-resistant EF-2 (class IIb), behaved codominantly in somatic cell hybrids. There was no complementation in hybrids formed between the two resistant mutants. The mutant parents and their hybrids, except those formed by fusion of CH-RE1.32 and wild-type cells, grew in the presence of toxin. To explain these results we suggest that CHO-K1 cells possess two functional copies of the gene for EF-2 and that CH-RE1.22c and CH-RE1.32 represent the homozygous (R/R) and heterozygous (R/S) states of resistance at the EF-2 gene locus. The failure of hybrids formed between CH-RE1.32 and wild-type cells to grow in toxin is a gene dosage effect. Codominant class IIa translational resistance is a selectable marker for the isolation of hybrids. It can be combined with a second, recessive, marker to provide a cell which is a "universal hybridizer" (10).     

Statistical analysis of an RNA titration series evaluates microarray precision and sensitivity on a whole-array basis

Background  

Concerns are often raised about the accuracy of microarray technologies and the degree of cross-platform agreement, but there are yet no methods which can unambiguously evaluate precision and sensitivity for these technologies on a whole-array basis.     

Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Differential binding of cyclic adenosine 3'' ,5''-monophosphate to the cyclic adenosine 3'' ,5''-monophosphate receptor protein in Escherichia coli.

Binding of cyclic adenosine 3' ,5'-monophosphate (cAMP) by the cAMP receptor protein in crude cell-free extracts of Escherichia coli was characterized. When cell were grown in glucose, binding was inhibited 50% relative to extracts from cells grown with succinate as carbon source . This inhibition could be relieved by dialysis.     

Determination of the optimal conditions for the continuous culture of Candida utilis in sugarcane stillage

Summary Candida utilis was continuously cultured in sugarcane stillage without any supplementation, but the yeast utilized the stillage nitrogen only partially. As a result, the cell biomass production was low and the residual chemical oxygen demand (COD), as well as the total nitrogen content of the effluent were high. The addition of 2–4 g/l ammonium phosphate and small amounts of Fe²⁺, Cu²⁺, and Zn²⁺, markedly enhanced both the everall nitrogen uptake and cell biomass production, decreased COD, and a higher critical dilution rate was attained. No substantial differences were found when pH was kept at 5.0 or allowed to evolve freely. The optimal temperature range was between 30 and 35°C.     

The 3-D structure of HIV-1 proteinase and the design of antiviral agents for the treatment of AIDS

A proteinase is essential for replication of HIV. Cloning and chemical synthesis have provided a sufficient supply of HIV-1 proteinase for the determination of its three-dimensional structure. Analogies between the structures of HIV-1 proteinase and the mammalian enzyme renin, which is involved in the control of blood pressure, have given important clues concerning the design of specific inhibitors that have antiviral activity.     

PKCλ/ι signaling promotes triple-negative breast cancer growth and metastasis

Triple-negative breast cancer (TNBC) is a distinct breast cancer subtype defined by the absence of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2/neu), and the patients with TNBC are often diagnosed with higher rates of recurrence and metastasis. Because of the absence of ER, PR and HER2/neu expressions, TNBC patients are insensitive to HER2-directed and endocrine therapies available for breast cancer treatment. Here, we report that expression of atypical protein kinase C isoform, PKCλ/ι, significantly increased and activated in all invasive breast cancer (invasive ductal carcinoma or IDC) subtypes including the TNBC subtype. Because of the lack of targeted therapies for TNBC, we choose to study PKCλ/ι signaling as a potential therapeutic target for TNBC. Our observations indicated that PKCλ/ι signaling is highly active during breast cancer invasive progression, and metastatic breast cancers, the advanced stages of breast cancer disease that developed more frequently in TNBC patients, are also characterized with high levels of PKCλ/ι expression and activation. Functional analysis in experimental mouse models revealed that depletion of PKCλ/ι significantly reduces TNBC growth as well as lung metastatic colonization. Furthermore, we have identified a PKCλ/ι-regulated gene signature consisting of 110 genes, which are significantly associated with indolent to invasive progression of human breast cancer and poor prognosis. Mechanistically, cytokines such as TGFβ and IL1β could activate PKCλ/ι signaling in TNBC cells and depletion of PKCλ/ι impairs NF-κB p65 (RelA) nuclear localization. We observed that cytokine-PKCλ/ι-RelA signaling axis, at least in part, involved in modulating gene expression to regulate invasion of TNBC cells. Overall, our results indicate that induction and activation of PKCλ/ι promote TNBC growth, invasion and metastasis. Thus, targeting PKCλ/ι signaling could be a therapeutic option for breast cancer, including the TNBC subtype.Breast cancer is a clinically heterogeneous disease and both intra and inter-tumor heterogeneities provide great challenges for developing successful therapies. Expressions (or absence thereof) of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2)/neu are widely used to clinically classify breast tumors into multiple therapeutic groups.¹ The ER/PR-positive and the HER2-positive breast cancer patients could be benefited from endocrine and HER2-targeted therapies.¹ However, triple-negative breast cancers (TNBCs), which represent ∼12–17% of all breast cancer,² lack ER, PR and HER2/neu expressions² and are not responsive to therapies targeting these receptors. Therefore, the only systemic therapy available for TNBC is chemotherapy.³ Furthermore, TNBC is associated with aggressive pathologic features like higher histology grade and mitotic index⁴ and often found to be associated with higher rate of metastasis and recurrence leading to limited clinical outcome.^{5, 6, 7, 8} Recurrence of TNBC tends to recur within a few years after successful initial treatment^{6, 9} and often develops metastasis to the bone, brain and lungs with poor prognosis.^{2, 6} Thus, identification of signaling pathways that regulate malignant progression of breast cancer subtypes, especially TNBCs, would be therapeutically important.In recent years, PKC signaling has been implicated in modulating invasion and metastasis of multiple tumors.^{10, 11} The PKC family consists of multiple serine/threonine kinases and the relative contribution of individual PKC isoforms during cancer progression varies due to pleiotropism.¹² PKC isoforms regulate diverse cellular functions such as cell-cycle regulation, cellular survival, cell–cell communications and apoptosis.¹³ In particular, atypical PKC isoforms, PKCζ and atypical protein kinase C lamda/iota (PKCλ/ι), are known to be important for chemotaxis, cell polarity, migration and wound healing processes.^{14, 15} Aberrations in all these processes are manifested in tumor progression and metastasis.¹⁴ Consistent with these notions, recent studies indicated that atypical PKCs are associated with various human cancers.^{10, 11} Importantly, the PKCλ/ι gene is located at the 3q26.2 genomic region, which is most frequently amplified in human cancer^{16, 17}, and overexpression of PKCλ/ι has been implicated in cancer development in multiple tissues including the lung,^{18, 19} pancreas,²⁰ stomach,²¹ colon,²² esophagus,²³ liver,²⁴ bile duct,²⁵ ovary,¹⁷ prostate²⁶ and brain.²⁷ Recently, few studies have been reported higher expression of PKCλ/ι in ER/PR- and HER-positive breast cancer and also in lymph node metastases.^{28, 29} Kojima et. al.²⁸ showed that PKCλ/ι expression is highly induced in the ER/PR- and HER2-positive IDCs compared with ductal carcinoma in situ (DCIS) and normal breast. PKCλ/ι forms apical-junctional complexes (AJCs) with other polarity proteins such as partitioning defective 3 homolog (PAR3) and partitioning defective 6 homolog (PAR6),^{30, 31, 32, 33} and invasiveness of breast tumor cells was shown to be associated with loss of PKCλ/ι localization from their apical domains.²⁸ In addition, predominant nuclear localization of PKCλ/ι in both normal and atypical ductal hyperplasia (ADH) lesions prompted the concept that PKCλ/ι might be in an inactive state in these lesions.²⁸ However, expression and activation of PKCλ/ι in TNBCs and the functional importance of PKCλ/ι signaling in relation to invasive breast cancer progression and metastasis are very poorly understood.^{10, 11}Here, we studied PKCλ/ι signaling during invasive progression of TNBC. We utilized expression evaluations in triple-negative IDCs as well as metastatic breast cancers of human patients, in vitro and in vivo functional assays, and global gene expression analysis of human patient samples. We concluded that PKCλ/ι signaling is an important regulator for invasion and metastatic progression of human breast cancers including triple-negative subtypes.     

Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli

Two genetic procedures were used to obtain amino acid replacements in the lacZ-encoded beta-galactosidase in Escherichia coli. Amino acid replacements could be obtained without regard to their effects on lactase activity by selecting spontaneous mutations that relieved the strong polarity of six nonsense mutations. When streaked on MacConkey- lactose indicator plates, approximately 75% of these mutants gave strong red lactose-fermenting colonies, and 25% gave white nonfermenting colonies. Mutants from 11 other nonsense codons were isolated directly using MacConkey-lactose indicator plates, on which positive color indication requires only 0.5% of the wildtype lactase activity. Among the total of 17 codons, 25 variant beta-galactosidases were identified using electrophoresis and thermal denaturation studies. The fitness effects of these variant beta-galactosidases were determined using competition experiments conducted with lactose as the sole nutrient limiting the growth rate in chemostat cultures. Three of the replacements were deleterious, one was selectively advantageous, and the selective effects of the remaining 21 were undetectable under conditions in which the smallest detectable selection coefficient was approximately 0.4%/generation.      

The small GTPase Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes.

Apoptosis plays an important role in regulating development and homeostasis of the immune system, yet the elements of the signaling pathways that control cell death have not been well defined. When expressed in Jurkat T cells, an activated form of the small GTPase Cdc42 induces cell death exhibiting the characteristics of apoptosis. The death response induced by Cdc42 is mediated by activation of a protein kinase cascade leading to stimulation of c-Jun amino terminal kinase (JNK). Apoptosis initiated by Cdc42 is inhibited by dominant negative components of the JNK cascade and by reagents that block activity of the ICE protease (caspase) family, suggesting that stimulation of the JNK kinase cascade can lead to caspase activation. The sequence of morphological events observed typically in apoptotic cells is modified in the presence of activated Cdc42, suggesting that this GTPase may account for some aspects of cytoskeletal regulation during the apoptotic program. These data suggest a means through which the biochemical and morphological events occurring during apoptosis may be coordinately regulated.