Development and Validation of a UPLC-MS/MS Method to Monitor Cephapirin Excretion in Dairy Cows following Intramammary Infusion

Cephapirin, a cephalosporin antibiotic, is used by the majority of dairy farms in the US. Fecal and urinary excretion of cephapirin could introduce this compound into the environment when manure is land applied as fertilizer, and may cause development of bacterial resistance to antibiotics critical for human health. The environmental loading of cephapirin by the livestock industry remains un-assessed, largely due to a lack of appropriate analytical methods. Therefore, this study aimed to develop and validate a cephapirin quantification method to capture the temporal pattern of cephapirin excretion in dairy cows following intramammary infusion. The method includes an extraction with phosphate buffer and methanol, solid-phase extraction (SPE) clean-up, and quantification using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The LOQ values of the developed method were 4.02 µg kg⁻¹ and 0.96 µg L⁻¹ for feces and urine, respectively. This robust method recovered >60% and >80% cephapirin from spiked blank fecal and urine samples, respectively, with acceptable intra- and inter-day variation (<10%). Using this method, we detected trace amounts (µg kg⁻¹) of cephapirin in dairy cow feces, and cephapirin in urine was detected at very high concentrations (133 to 480 µg L⁻¹). Cephapirin was primarily excreted via urine and its urinary excretion was influenced by day (P = 0.03). Peak excretion (2.69 mg) was on day 1 following intramammary infusion and decreased sharply thereafter (0.19, 0.19, 0.08, and 0.17 mg on day 2, 3, 4, and 5, respectively) reflecting a quadratic pattern of excretion (Quadratic: P = 0.03). The described method for quantification of cephapirin in bovine feces and urine is sensitive, accurate, and robust and allowed to monitor the pattern of cephapirin excretion in dairy cows. This data will help develop manure segregation and treatment methods to minimize the risk of antibiotic loading to the environment from dairy farms.     

Detection of a calmodulin-sensitive cyclic nucleotide phosphodiesterase in rat parotid gland

Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca²⁺ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca²⁺ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin.     

Synapsis of attachment sites during lambda integrative recombination involves capture of a naked DNA by a protein-DNA complex

During lambda integration, Int recombinase must specifically bind to and cut attachment sites on both the viral and host chromosomes. We show here by foot-printing and by a novel cleavage assay that the bacterial attachment site, attB, cannot stably bind Int in competition with other DNAs. Instead, during recombination reactions, attB obtains its Int by collision with the intasome, a nucleoprotein assembly that forms on the viral attachment site, attP. Our cleavage assay also shows that the capture of attB by the attP intasome does not depend on DNA homology between the two sites; synapsis is governed solely by protein-protein and protein-DNA interactions.     

B-lymphocyte responses in the large intestine and mesenteric lymph nodes of mice infected with Eimeria falciformis (Apicomplexa)

B-cell responses of 3 immunoglobulin isotypes (IgA, IgG, and IgM) were investigated in the large intestine and mesenteric lymph nodes (MLN) of naive or immune mice after inoculation of oocysts of Eimeria falciformis. Primary and anamnestic IgA and IgG lymphocyte responses to E. falciformis occurred in the large intestine of nonimmune and immune mice, respectively. IgA-containing lymphocytes (IgAc) were the largest population of responding B cells in the large intestine. In infected mice, IgAc accumulated in the apical portion of the lamina propria, whereas IgG-containing lymphocytes (IgGc) were more numerous at the base of the lamina propria. No significant increase in the number of IgM-containing lymphocytes (IgMc) was observed in the lamina propria of the large intestine. Primary but no anamnestic B-cell responses occurred in the MLN, and immune mice actually had reduced numbers of IgAc and IgGc in the MLN when compared with naive mice. IgGc were the largest population of responding B cells in the MLN. Thus, IgAc appear to accumulate preferentially at the site of parasite development, whereas IgGc are primarily localized deeper in the lamina propria of the large intestine and in the draining lymph nodes of mice infected with E. falciformis.     

Heterogeneity of Lethals in a "Simple" Lethal Complementation Group

Of 24 ethyl methanesulphonate-induced, recessive-lethal mutations in the region 9E1-9F13 of the X chromosome of Drosophila melanogaster, eight fall into a typically homogeneous lethal complementation group associated with the raspberry (ras) locus. Mutations in this group have previously been shown to be pleiotropic, affecting not only ras but also two other genetic entities, gua 1 and pur 1, which yield auxotrophic mutations.--The eight new mutations have been characterized phenotypically in double heterozygotes with gua 1, pur 1 and ras mutations. Despite their homogeneity in lethal complementation tests, the mutations prove quite diverse. For example, two mutations have little or no effect on eye color in double heterozygotes with ras2. The differences between the lethals are allele-specific and cannot be explained as a trivial outcome of a hypomorphic series.--Taken alone, the lethal complementation studies mask the complexity of the locus and the diversity of its recessive lethal alleles. By extension, we argue that the general use of lethal saturation studies provides an unduly simplified image of genetic organization. We suggest that the reason why recessive lethal mutations rarely present complex complementation patterns is that complex loci tend to produce mutations that affect several subfunctions.     

Lack of cellular cytotoxicity by human mononuclear cells to Giardia

Cytotoxicity of mononuclear leukocytes (MNL) to Giardia lamblia was compared by using two techniques. The first method assessed the viability of surviving Giardia directly by culturing and the second method measured release of incorporated [3H] thymidine. Cytotoxicity, as measured directly by culturing and visual assessment, showed that the numbers of surviving Giardia decreased over time whether cultured with or without MNL but that Giardia survived significantly better in the presence of MNL at 18 hr (21.8 +/- 8.3% with MNL compared with 3.4 +/- 1.5% without MNL). Release of [3H]thymidine increased whether Giardia were cultured with or without MNL and although there was a tendency for increased release with MNL, there was no significant difference. Dead labeled Giardia released significantly more label in the presence of MNL than without MNL, suggesting that MNL cause release of [3H]thymidine after phagocytosis. The thymidine release assay therefore does not measure spontaneous cytotoxicity of MNL to Giardia.