No abstract

1. The physiological effects of two Phe-Met-Arg-Phe-NH2 (FMRFamide)-related neuropeptides isolated from the free-living nematode Panagrellus redivivus, SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2), were examined using neuromuscular preparations from the parasitic nematode Ascaris suum. 2. PF1 and PF2 hyperpolarized muscle membrane and induced sustained flaccid paralysis, independent of external Cl-, in both innervated and denervated preparations. 3. PF1 reversed spastic contractions induced by the cholinomimetic levamisole, elevated K+, or the excitatory nematode FMRFamide-related neuropeptides KNEFIRFamide or KHEYLRFamide. 4. PF1 reversal of levamisole contraction was blocked by pretreatment with agents that interfere with nitric oxide (NO) synthesis (e.g., N-nitro-L-arginine), whereas sodium nitroprusside, which releases NO in solution, mimicked PF1 and PF2. 5. NO synthase activity, monitored by the conversion of [3H]arginine to [3H]citrulline, was twice as abundant in A. suum hypodermis as in muscle, but was not present in reproductive tissue. The relative abundance of NO synthase activity in these tissues was similar to the observed specific binding of [3H]PF1. 6. These results suggest that the inhibitory effects of PF1 and PF2 on nematode somatic muscle are mediated by NO, and that the hypodermis may serve a role in this process analogous to that of the endothelium in vertebrate vasculature.

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Clonally derived recombinant cell lines are highly desired to achieve consistent production of recombinant biotherapeutics. Despite repeated rounds of cloning by limiting dilution or single cell cloning, the resulting cell lines have often been observed to diverge, becoming a heterogeneous population and losing productivity over long-term sub-culturing. To understand the underlying molecular mechanisms, we developed quantitative polymerase chain reaction (qPCR) assays for the analysis of transgene copy number distribution in single recombinant cells isolated from Chinese hamster ovary (CHO) cell lines. Single cells were obtained by fluorescence activated cell sorting (FACS) technology and lysed directly in 96-well plates. qPCR assays were then applied to analyze the quantity and distribution of transgenes in those single cells. Results revealed multiple types of transgene copy number distribution profiles from those clonally derived CHO cell lines. The cell lines that maintained productivity over time displayed relatively constant and homogeneous transgene copy number distributions; while most of those cell lines exhibiting a loss of productivity over time showed varying degrees of transgene copy number heterogeneity and distribution drift with passaging. Some cell lines showed the existence of a significant portion of cells lacking the transgenes (referred to as negative cells in this study) and the percentage of those negative cells increased with subsequent generations. Criteria based on transgene copy number distribution profiles were developed to assess cell line suitability for clinical applications, which include (i) percentage of negative cells; (ii) standard deviation of qPCR threshold cycle (C(t) ) value, a measure of population heterogeneity; (iii) mean C(t) changes during aging, a measure of population drift. By implementing these criteria, undesirable cell lines were eliminated for further clinical and commercial applications.