On the efficacy of commonly used ribonuclease inhibitors.

The ability of a number of commonly used inhibitors to inhibit pancreatic ribonuclease has been studied. At ribonuclease concentrations of 10 or 100 g/ml, heparin, polyvinylsulfate and proteinase K, at concentrations reported for their use in the literature, were ineffective in inhibiting RNase digestion of ³H-uridine labelled RNA from $\text{Streptomyces antibioticus}$. In contrast, macaloid, diethylpyrocarbonate and sodium dodecyl sulfate were all effective inhibitors, with the degree of effectiveness decreasing in the order stated. Further, at inhibitor concentrations which allowed RNase conversion of only 50% of the labelled RNA to acid soluble products, a larger percentage of the acid insoluble digestion products sedimented in the “high molecular weight” range (4–16s) when macaloid was the inhibitor used than when diethylpyrocarbonate was the inhibitor.     

A functional analysis of N-glycosylation-related genes on sialylation of recombinant erythropoietin in six commonly used mammalian cell lines

Significant efforts have been made to improve the sialylation of recombinant glycoproteins with the aim of extending their in vivo circulation time. Here, we report a systematic functional analysis of 31 N-glycosylation-related genes on sialylation of recombinant EPO in six cell lines. BHK and CHO cells were found to sialylate recombinant EPO most effectively. None of the 31 genes, individually or in combination, was able to improve EPO sialylation in these cells. HEK293, Cos-7 and 3T3 cells showed intermediate sialylation capabilities, whereas NS0 cells sialylated recombinant EPO poorly. Overexpression of ST6GalI, ST3GalIII or ST3GalIV, but not ST3GalVI, was able to improve EPO sialylation in these four cell lines. qRT-PCR experiments revealed that ST3GalIII and ST3GalIV are indeed under expressed in HEK293, 3T3 and NS0 cells. Co-expression of upstream glycogenes failed to synergize with these sialyltransferases to further enhance sialylation, suggesting that the upstream glycogenes are all expressed at sufficient levels.     

Mu receptor binding of some commonly used opioids and their metabolites.

The binding affinity to the mu receptor of some opioids chemically related to morphine and some of their metabolites was examined in rat brain homogenates with 3H-DAMGO. The chemical group at position 6 of the molecule had little effect on binding (e.g. morphine-6-glucuronide Ki = 0.6 nM; morphine = 1.2 nM). Decreasing the length of the alkyl group at position 3 decreased the Ki values (morphine less than codeine less than ethylmorphine less than pholcodine). Analgesics with high clinical potency containing a methoxyl group at position 3 (e.g. hydrocodone, Ki = 19.8 nM) had relatively weak receptor binding, whilst their O-demethylated metabolites (e.g. hydromorphone, Ki = 0.6 nM) had much stronger binding. Many opioids may exert their pharmacological actions predominantly through metabolites.     

Cytogenetic harvesting of commonly used tumor cell lines

Tumor cell lines are widely used both as disease models and, increasingly, as genomic resources for the ascertainment of new cancer genes. Cytogenetic analysis remains a major route to uncovering the cancer genome. However, cancer cell lines vary inexplicably in their harvesting preferences, which must, therefore, be determined by trial and error. This article describes harvesting protocols optimized empirically for 550 commonly used, mainly human, cancer cell lines together with evidence-based procedures to assist in determining conditions for unlisted cell lines and subsidiary protocols for cytogenetic analysis using G-banding and fluorescence in situ hybridization.     

Cytotoxicity of commonly used solvents at elevated temperatures

At 43 degrees C (but not at 41 degrees C), organic solvents used to dissolve water-insoluble chemotherapeutic agents become themselves lethal to cells. This finding is not unique to Chinese hamster cells (HA-1); mouse mammary sarcoma cells (EMT-6) behave similarly. The solvent concentrations involved are in the range of those needed to make drug solutions. Hence experiments measuring drug-cell interactions at elevated temperatures must include controls which independently measure solvent effects.     

Structural specificities of five commonly used DNA nucleases

Five commonly used nucleases were surveyed for their ability to distinguish among several different DNA backbone configurations. The digestion data suggest that: (1) DNAase I binds across the minor groove; whereas (2) nuclease S1 and (3) micrococcal nuclease bind to an exposed single strand; (4) copper/phenanthroline seeks a base-pair step; and (5) DNAase II requires just a stacked single strand of limited exposure. Only micrococcal nuclease is demonstrably base-specific, with a strong preference for T, A over C, G in any structural context.     

Physical properties and compact analysis of commonly used direct compression binders

This study investigated the basic physico-chemical property and binding functionality of commonly used commercial direct compression binders/fillers. The compressibility of these materials was also analyzed using compression parameters derived from the Heckel, Kawakita, and Cooper-Eaton equations. Five classes of excipients were evaluated, including microcrystalline cellulose (MCC), starch, lactose, dicalcium phosphate (DCP), and sugar. In general, the starch category exhibited the highest moisture content followed by MCC, DCP, lactose, and finally sugars; DCP displayed the highest density, followed by sugar, lactose, starch, and MCC; the material particle size is highly processing dependent. The data also demonstrated that MCC had moderate flowability, excellent compressibility, and extremely good compact hardness; with some exceptions, starch, lactose, and sugar generally exhibited moderate flowability, compressibility, and hardness; DCP had excellent flowability, but poor compressibility and hardness. This research additionally confirmed the binding mechanism that had been well documented: MCC performs as binder because of its plastic deformation under pressure; fragmentation is the predominant mechanism in the case of lactose and DCP; starch and sugar perform by both mechanism.     

Antigenic features of protein carriers commonly used in immunisation trials

An aluminium hydroxide adjuvant induced a more elevated and rapid immune responses against short peptides conjugated to the Keyhole Lympet Hemocyanin carrier than immuneasy adjuvant. Furthermore, since carrier proteins may compete with the fused or chemically linked polypeptides in eliciting antigen-specific immune response, we classified the immunogenicity of the most common carrier proteins used in molecular biology for antigen expression and mouse immunisation. The disulfide isomerase protein A gave a carrier with the lowest immunogenicity whilst disulfide isomerase protein C gave the highest immunogenicity and therefore should be avoided as a fusion partner. Using this protein as a model, we identified and located the immunodominant epitopes along its sequence. These results now enable the combination of carrier and immunisation conditions to be optimized.     

几种常用防腐剂对日化产品中腐败微生物抑制效果研究

了解几种常用防腐剂在日化产品基质中的防腐效果。采用最低抑菌浓度方法测试卡松、对羟基苯甲酸甲酯、苯氧乙醇、咪唑烷基脲、苯甲醇对腐败微生物的抑菌活性;采用微生物挑战性实验验证卡松、对羟基苯甲酸甲酯、咪唑烷基脲、苯氧乙醇和苯甲醇的抑菌效果。日化产品中分离的腐败微生物对卡松耐受性接近0.0015%,接近《化妆品卫生规范》2007版的限定,其它4种防腐剂在规定的使用范围内对分离的微生物都具有较好的抑制效果;卡松剂量在0.0015%、对羟基苯甲酸甲酯剂量在0.2%、咪唑烷基脲剂量在0.075%、苯甲醇剂量在0.2%时日化产品能通过微生物挑战。日化企业应根据实际情况筛选、复配防腐剂,建立更加高效、经济的防腐体系。