Organization handbook for Minnesota rural youth groups

24 pages; includes illustrations and photographsThis archival publication may not reflect current scientific knowledge or recommendations. Current information available from the University of Minnesota Extension: https://www.extension.umn.edu

Phosphorylation and Transport in the Na-K-2Cl Cotransporters, NKCC1 and NKCC2A, Compared in HEK-293 Cells

Na-K-2Cl cotransporters help determine cell composition and volume. NKCC1 is widely distributed whilst NKCC2 is only found in the kidney where it plays a vital role reabsorbing 20% of filtered NaCl. NKCC2 regulation is poorly understood because of its restricted distribution and difficulties with its expression in mammalian cell cultures. Here we compare phosphorylation of the N-termini of the cotransporters, measured with phospho-specific antibodies, with bumetanide-sensitive transport of K+ (86Rb+) (activity) in HEK-293 cells stably expressing fNKCC1 or fNKCC2A which were cloned from ferret kidney. Activities of transfected transporters were distinguished from those of endogenous ones by working at 37°C. fNKCC1 and fNKCC2A activities were highest after pre-incubation of cells in hypotonic low-[Cl−] media to reduce cell [Cl−] and volume during flux measurement. Phosphorylation of both transporters more than doubled. Pre-incubation with ouabain also strongly stimulated fNKCC1 and fNKCC2A and substantially increased phosphorylation, whereas pre-incubation in Na+-free media maximally stimulated fNKCC1 and doubled its phosphorylation, but inhibited fNKCC2A, with a small increase in its phosphorylation. Kinase inhibitors halved phosphorylation and activity of both transporters whereas inhibition of phosphatases with calyculin A strongly increased phosphorylation of both transporters but only slightly stimulated fNKCC1 and inhibited fNCCC2A. Thus kinase inhibition reduced phosphorylation and transport, and transport stimulation was only seen when phosphorylation increased, but transport did not always increase with phosphorylation. This suggests phosphorylation of the N-termini determines the transporters' potential capacity to move ions, but final activity also depends on other factors. Transport cannot be reliably inferred solely using phospho-specific antibodies on whole-cell lysates

Bi-allelic Mutations in NDUFA6 Establish Its Role in Early-Onset Isolated Mitochondrial Complex I Deficiency.

Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects' fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects' fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis

The argan - multipurpose tree of Morocco

Joseph Hooker and John Ball, writing of Argania spinosa (L.) Skeels, said that "this tree is rightly regarded as the most interesting vegetable product of Marocco, being confined to that empire and to a very circumscribed area in it, belonging to an almost exclusively tropical family, yielding a most important article of diet to the inhabitants, and a wood that for hardness and durability rivals any hitherto described" (Hooker & Ball, 1878). More than a century later the argan continues to be of manifold interest; to the botanist and ecologist because of its apparent Moroccan endemism and its disjunct distribution in relation to its family, the Sapotaceae; to the food technologist because of its highly nutritious oil whose untapped commercial potential is hindered by problems with its extraction; and to foresters and local inhabitants because of its central role as a stabilizer of soil and as a source of fuel and food for goats. There must now, however, be added a further interest in the argan: a grave concern for its future