Vesicle endocytosis is essential for maintaining synaptic transmission. Its key step, membrane scission, is thought to be mediated by the GTPase dynamin in all forms of endocytosis at synapses. Our findings indicate that GTP-independent and probably dynamin-independent endocytosis co-exist with GTP- and dynamin-dependent endocytosis at the same synaptic nerve terminal, the calyx of Held, in rats. This previously undescribed form of endocytosis could be slow (tens of seconds) and/or rapid (a few seconds), similar to GTP- and dynamin-dependent endocytosis. It was activated during intense stimulation, whereas GTP- and dynamin-dependent endocytosis dominated during mild stimulation. These results establish a new model, in which vesicles are divided into two pools depending on their requirement for GTP and dynamin for retrieval. The GTP- and dynamin-dependent pool has higher priority for release and retrieval, but limited capacity, saturation of which leads to release and thus retrieval of GTP- and dynamin-independent vesicles.

ALDH3A1 (aldehyde dehydrogenase 3A1) is abundant in the mouse cornea but undetectable in the lens, and ALDH1A1 is present at lower (catalytic) levels in the cornea and lens. To test the hypothesis that ALDH3A1 and ALDH1A1 protect the anterior segment of the eye against environmentally induced oxidative damage, Aldh1a1(؊/؊)/Aldh3a1(؊/؊) double knock-out and Aldh1a1(؊/؊) and Aldh3a1(؊/؊) single knock-out mice were evaluated for biochemical changes and cataract formation (lens opacification). The Aldh1a1/Aldh3a1-and Aldh3a1-null mice develop cataracts in the anterior and posterior subcapsular regions as well as punctate opacities in the cortex by 1 month of age. The Aldh1a1-null mice also develop cataracts later in life (6 -9 months of age). One-to three-month-old Aldh-null mice exposed to UVB exhibited accelerated anterior lens subcapsular opacification, which was more pronounced in Aldh3a1(؊/؊) and Aldh3a1(؊/؊)/Aldh1a1(؊/؊) mice compared with Aldh1a1(؊/؊) and wild type animals. Cataract formation was associated with decreased proteasomal activity, increased protein oxidation, increased GSH levels, and increased levels of 4-hydroxy-2-nonenal-and malondialdehyde-protein adducts. In conclusion, these findings support the hypothesis that corneal ALDH3A1 and lens ALDH1A1 protect the eye against cataract formation via nonenzymatic (light filtering) and enzymatic (detoxification) functions.

The results suggest that, under oxidative stress, HNE produced in the lens epithelium can cause toxicity and thus contribute to oxidation-induced cataractogenesis. Furthermore, the studies indicate that ALDH1A1 is a critical isozyme for maintaining clarity in human, rat, and mouse lenses.

We have constructed an ALDH3a1 null mouse to investigate the role of this enzyme that comprises nearly one-half of the total water-soluble protein in the mouse corneal epithelium. ALDH3a1-deficient mice are viable and fertile, have a corneal epithelium with a water-soluble protein content approximately half that of wild-type mice, and contain no ALDH3a1 as determined by zymograms and immunoblots. Despite the loss of protein content and ALDH3a1 activity, the ALDH3a1 ؊/؊ mouse corneas appear indistinguishable from wild-type corneas when examined by histological analysis and electron microscopy and are transparent as determined by light and slit lamp microscopy. There is no evidence for a compensating protein or enzyme. Even though the function of ALDH3a1 in the mouse cornea remains unknown, our data indicate that its enzymatic activity is unnecessary for corneal clarity and maintenance, at least under laboratory conditions.

Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome. The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in XEMBL3. Genomic maps were drawn for three ORS571 Nif gene loci. Forty- Rhizobium sp. strain ORS571, uniquely among characterized members of the family Rhizobiaceae, conducts N2 fixation during active bacterial growth (16) as well as during plant nodule symbiosis, in which it is a nonproliferating endosymbiont. ORS571 forms classical, synchronous nodules in roots and in stem lateral root primordia with the host leguminous plant Sesbania rostrata (32). As such, ORS571 appears to be a chimeric N2-fixing bacterium with properties of both orthodox rhizobia and diazotrophic bacteria. Operationally, the chimeric nature of ORS571 N2 fixation facilitates a genetic analysis of rhizobial N2 fixation processes because ORS571 N2 fixation-defective (Nif) mutants may be directly selected and screened ex planta.We have recently developed the vector-insertion (Vi) mutagenesis-cloning strategy and have applied it to ORS571 (9). This method allows the direct molecular cloning of any ORS571 genes in which mutants can be selected or screened. Vi mutagenesis-cloning consists of the following protocol. (Table 1). ORS571 recipients carrying ISSOR-mediated plasmid-genome cointegrates were obtained after platings on rich media containing kanamycin and streptomycin as described previously (9). From this collection, candidate strains were screened by replica plating on ORSNif medium, a defined, twofold-diluted M9 minimal medium containing 0.4% D-glucose, 0.4% succinic acid titrated to pH 6.0 with KOH, 1 mM MgSO4, 0.5 mM CaCl2, 1 ,ug of FeCl3 per ml, 1 ,ug of NaMoO4 per ml, 1 ,ug of pantothenate per ml, 1 ,ug of nicotinate per ml, and 0.3 jig of D-biotin per ml, with and without 15 mM (NH4)2SO4 added as a nitrogen source. Plates were solidified with 0.85% agarose (Clonetech) and incubated in a growth chamber at 30°C under a continous flow atmosphere of 98% N2-1% C0O-1% 02-ORS571 Nif::Vi mutants were identified by their inability to utilize N2 as a nitrogen source on solid media (9). Putative 72 on May 11, 2018 by guest Tn5 is first introduced into