Positive immunolabelling for feline infectious peritonitis in an African lion (Panthera leo) with bilateral panuveitis.

A 15-year-old male African lion (Panthera leo) was presented with blindness due to bilateral panuveitis with retinal detachment. Feline coronavirus (FCoV) antigen was identified immunohistochemically in ocular macrophages, consistent with a diagnosis of feline infectious peritonitis (FIP) infection. This is the first report of FIP in an African lion and the first report of ocular FIP in a non-domestic felid.

Concurrent infections of Fasciola, Schistosoma and Amphistomum spp. in cattle from Kafue and Zambezi river basins of Zambia.

This study investigated interactions among Fasciola gigantica, Schistosoma spp. and Amphistomum spp. concurrent natural infections in Zambian cattle, based on egg and worm counts. In the abattoir 315 cattle were screened for worms of F. gigantica in the liver, Schistosoma spp. in mesenteric veins and/or Amphistomum spp. in the rumen. One hundred and thirty-three (42.2%) of the abattoir-examined cattle harboured one, two or all three trematodes. Of 133 cattle, 50 were randomly selected for worm and egg counts. The mean numbers (+/- SD) of Amphistomum, Schistosoma and Fasciola were 622.08 (+/- 97.87), 33.68 (+/- 7.44) and 19.46 (+/- 4.58), respectively. A total of 32% harboured all the three trematodes, 66% had F. gigantica and Amphistomum spp. infections, 52% had Schistosoma spp. and Amphistomum spp. infections while 32% had F. gigantica and Schistosoma infections. A positive correlation (P = 0.014) was found between F. gigantica and Amphistomum worm burdens. There were no correlations between Amphistomum and Schistosoma worm burdens and between F. gigantica and Schistosoma worm burdens. It may be concluded that there is no significant cross-protection among these trematodes in cattle in endemic areas.

Selective accumulation of the high molecular weight neurofilament subunit within the distal region of growing axonal neurites.

Axonal maturation in situ is accompanied by the transition of neurofilaments (NFs) comprised of only NF-M and NF-L to those also containing NF-H. Since NF-H participates in interactions of NFs with each other and with other cytoskeletal constituents, its appearance represents a critical event in the stabilization of axons that accompanies their maturation. Whether this transition is effected by replacement of "doublet" NFs with "triplet" NFs, or by incorporation of NF-H into existing doublet NFs is unclear. To address this issue, we examined the distribution of NF subunit immunoreactivity within axonal cytoskeletons of differentiated NB2a/d1 cell and DRG neurons between days 3-7 of outgrowth. Endogenous immunoreactivity either declined in a proximal-distal gradient or was relatively uniform along axons. This distribution was paralleled by microinjected biotinylated NF-L. By contrast, biotinylated NF-H displayed a bipolar distribution, with immunoreactivity concentrated within the proximal- and distal-most axonal regions. Proximal biotinylated NF-H accumulation paralleled that of endogenous NF immunoreactivity; however, distal-most biotinylated NF-H accumulation dramatically exceeded that of endogenous NFs and microinjected NF-L. This phenomenon was not due to co-polymerization of biotin-H with vimentin or alpha-internexin. This phenomenon declined with continued time in culture. These data suggest that NF-H can incorporate into existing cytoskeletal structures, and therefore suggest that this mechanism accounts for at least a portion of the accumulation of triplet NFs during axonal maturation. Selective NF-H accumulation into existing cytoskeletal structures within the distal-most region may provide de novo cytoskeletal stability for continued axon extension and/or stabilization.

Neurofilaments consist of distinct populations that can be distinguished by C-terminal phosphorylation, bundling, and axonal transport rate in growing axonal neurites.

We examined the steady-state distribution and axonal transport of neurofilament (NF) subunits within growing axonal neurites of NB2a/d1 cells. Ultrastructural analyses demonstrated a longitudinally oriented "bundle" of closely apposed NFs that was surrounded by more widely spaced individual NFs. NF bundles were recovered during fractionation and could be isolated from individual NFs by sedimentation through sucrose. Immunoreactivity toward the restrictive C-terminal phospho-dependent antibody RT97 was significantly more prominent on bundled than on individual NFs. Microinjected biotinylated NF subunits, GFP-tagged NF subunits expressed after transfection, and radiolabeled endogenous subunits all associated with individual NFs before they associated with bundled NFs. Biotinylated and GFP-tagged NF subunits did not accumulate uniformly along bundled NFs; they initially appeared within the proximal portion of the NF bundle and only subsequently were observed along the entire length of bundled NFs. These findings demonstrate that axonal NFs are not homogeneous but, rather, consist of distinct populations. One of these is characterized by less extensive C-terminal phosphorylation and a relative lack of NF-NF interactions. The other is characterized by more extensive C-terminal NF phosphorylation and increased NF-NF interactions and either undergoes markedly slower axonal transport or does not transport and undergoes turnover via subunit and/or filament exchange with individual NFs. Inhibition of phosphatase activities increased NF-NF interactions within living cells. These findings collectively suggest that C-terminal phosphorylation and NF-NF interactions are responsible for slowing NF axonal transport.

The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation.

The forms in which neurofilament (NF) subunits undergo axonal transport is controversial. Recent studies from have provided real-time visualization of the slow axonal transport of NF subunits by transfecting neuronal cultures with constructs encoding green fluorescent protein (GFP)-conjugated NF-M subunits. In our studies in differentiated NB2a/d1 cells, the majority NF subunits underwent transport in the form of punctate NF precursors, while studies in cultured neurons have demonstrated transport of NF subunits in predominantly filamentous form. Although different constructs were used in these studies, transfection of the same cultured neurons with our construct yielded the filamentous pattern observed by others, while transfection of our cultures with their construct generated punctate structures, confirming that the observed differences did not reflect variances in assembly-competence among the constructs. Manipulation of intracellular kinase, phosphatase, and protease activities shifted the predominant form of GFP-conjugated subunits between punctate and filamentous, confirming, as shown previously for vimentin, that punctate structures represent precursors for intermediate filament formation. Since these prior studies were conducted at markedly differing neuronal differentiation states, we tested the alternate hypothesis that these differing results reflected developmental alterations in NF dynamics that accompany various stages of neuritogenesis. We conducted time-course analyses of transfected NB2a/d1 cells, including monitoring of transfected cells over several days, as well as transfecting cells at varying intervals prior to and following induction of differentiation and axonal neurite outgrowth. GFP-conjugated subunits were predominantly filamentous during the period of most robust axonal outgrowth and NF accumulation, and presented a mixed profile of punctate and filamentous forms prior to neuritogenesis and following the developmental slowing of neurite outgrowth. These analyses demonstrate that NF subunits are capable of undergoing axonal transport in multiple forms, and that the predominant form in which NF subunits undergo axonal transport varies in accord with the rate of axonal elongation and accumulation of NFs within developing axons.

Hypophosphorylated neurofilament subunits undergo axonal transport more rapidly than more extensively phosphorylated subunits in situ.

Axonal transport of neurofilaments (NFs) has long been considered to be regulated by phosphorylation. We present evidence that in optic axons of normal mice, the rate of NF axonal transport is inversely correlated with the NF phosphorylation state. In addition to 200 kDa NF-H and 145 kDa NF-M, axonal cytoskeletons from CNS contained a range of phospho-variants of NF-H migrating between 160-200 kDa, and of NF-M migrating at 97-145 kDa. While 160 kDa phospho-variants of NF-H have been well characterized, we confirmed the identity of the previously-described 97 kDa species as a hypophospho-variant of NF-M since (1) pulse-chase metabolic labeling confirmed the 97 kDa species to be a new synthesis product that was converted by phosphorylation over time into a form migrating at 145 kDa, (2) the 97 kDa protein reacted with multiple NF-M antibodies, including one specific for hypophosphorylated NF-M, and (3) dephosphorylation converted NF-M isoforms to 97 kDa. Autoradiographic analyses following metabolic radiolabeling demonstrated that hypophosphorylated NF-H and NF-M isoforms underwent substantially more rapid transport in situ than did extensively phosphorylated isoforms, while NF-H subunits bearing a developmentally delayed C-terminal phospho-epitope transported at a rate slower than that of total 200 kDa NF-H. Differential transport of phospho-variants also highlights that these variants are not homogeneously distributed among NFs, but are segregated to some extent among distinct, although probably overlapping, NF populations, indicating that axonal NFs are not homogeneous with respect to phosphorylation state.

Phospho-dependent association of neurofilament proteins with kinesin in situ.

Recent studies demonstrate co-localization of kinesin with neurofilament (NF) subunits in culture and suggest that kinesin participates in NF subunit distribution. We sought to determine whether kinesin was also associated with NF subunits in situ. Axonal transport of NF subunits in mouse optic nerve was perturbed by the microtubule (MT)-depolymerizing drug vinblastine, indicating that NF transport was dependent upon MT dynamics. Kinesin co-precipitated during immunoprecipitation of NF subunits from optic nerve. The association of NFs and kinesin was regulated by NF phosphorylation, since (1) NF subunits bearing developmentally delayed phospho-epitopes did not co-purify in a microtubule motor preparation from CNS while less phosphorylated forms did; (2) subunits bearing these phospho-epitopes were selectively not co-precipitated with kinesin; and (3) phosphorylation under cell-free conditions diminished the association of NF subunits with kinesin. The nature and extent of this association was further examined by intravitreal injection of (35)S-methionine and monitoring NF subunit transport along optic axons. As previously described by several laboratories, the wave of NF subunits underwent a progressive broadening during continued transport. The front, but not the trail, of this broadening wave of NF subunits was co-precipitated with kinesin, indicating that (1) the fastest-moving NFs were associated with kinesin, and (2) that dissociation from kinesin may foster trailing of NF subunits during continued transport. These data suggest that kinesin participates in NF axonal transport either by directly translocating NFs and/or by linking NFs to transporting MTs. Both Triton-soluble as well as cytoskeleton-associated NF subunits were co-precipitated with kinesin; these data are considered in terms of the form(s) in which NF subunits undergo axonal transport.

C-terminal phosphorylation of the high molecular weight neurofilament subunit correlates with decreased neurofilament axonal transport velocity.

We probed the relationship of NF axonal transport of neurofilaments (NFs) to their phosphorylation state by comparing these parameters in two closely-aged groups of young adult mice - 2 and 5 months of age. This particular time interval was selected since prior studies demonstrate that optic axons have already completed axonal caliber expansion and attained adult NF levels by 2 months but, as shown herein, continue to increase NF-H C-terminal phosphorylation. NF axonal transport was monitored by autoradiographic analysis of the distribution of radiolabeled subunits immunoprecipitated from optic axon segments at intervals following intravitreal injection of 35S-methionine. Both the peak and front of radiolabeled NFs translocated faster in 2- vs. 5-month-old mice. This developmental decline in NF transport rate was not due to reduced incorporation of NFs into the cytoskeleton, nor to an overall decline in slow axonal transport. By excluding or minimizing other factors, these findings support previous conclusions that C-terminal NF phosphorylation regulates NF axonal transport.

Kinesin-mediated transport of neurofilament protein oligomers in growing axons.

We examined cytoskeleton-associated forms of NF proteins during axonal neuritogenesis in cultured dorsal root ganglion (DRG) neurons and NB2a/d1 neuroblastoma. In addition to filamentous immunoreactivity, we observed punctate NF immunoreactivity throughout perikarya and neurites. Immuno-electron microscopy revealed this punctate immunoreactivity to consist of non-membrane-bound 75 nm round/ovoid structures consisting of amorphous, fibrous material. Endogenous and microinjected NF subunits incorporated into dots prior to their accumulation within filaments. A transfected GFP-conjugated NF-M incorporated into dots and translocated at a rate consistent with slow axonal transport in real-time video analyses. Some dots converted into a filamentous form or exuded filamentous material during transport. Dots contained conventional kinesin immunoreactivity, associated with microtubules, and their transport into axons was blocked by anti-kinesin antibodies and nocodazole. These oligomeric structures apparently represent one form in which NF subunits are transported in growing axons and may utilize kinesin as a transport motor.

Neurofilament subunits can undergo axonal transport without incorporation into Triton-insoluble structures.

We examined the form(s) in which NF subunits undergo axonal transport. Pulse-chase radiolabeling analyses with 35S-methioinine revealed that newly synthesized Triton-soluble NF subunits accumulated within axonal neurites elaborated by NB2a/d1 neuroblastoma prior to the accumulation of Triton-insoluble subunits. Gel chromatographic, immunological, ultrastructural, and autoradiographic analyses of Triton-soluble axonal fractions demonstrated that radiolabeled, Triton-soluble subunits were associated with NFs. Triton-soluble, radiolabeled axonal NF subunits were also detected within retinal ganglion cell axons following intravitreal injection of 35S-methioinine. Microinjected biotinylated subunits were prominent within axonal neurites of NB2a/d1 cells and cultured dorsal root ganglion neurons substantially before they were retained following Triton-extraction. Prevention of biotinylated subunit, but not dextran tracer, translocation into neurites by nocodazole confirmed that microinjected subunits did not enter axons merely due to diffusion or injection-based pressure. Immuno-EM confirmed the association of biotin label with axonal NFs. These findings point towards multiple populations of NF subunits within axons and leave open the possibility that axonal NFs may be more dynamic than previously considered.

Extensive phosphorylation and axonal transport of triton-soluble neurofilament subunits.

The low abundance of soluble neurofilament (NF) subunits in mature axons has suggested that newly synthesized NF proteins rapidly assemble into highly stable polymers and associate with the Triton X-100-insoluble cytoskeleton. Here we present evidence for multiple populations of NFs and NF subunits, distinguished by differential solubility in Triton, within perikarya and axons of neurons in situ and in culture. We further demonstrate, using microinjection of "tagged" NF subunits and by pulse-chase radiolabeling of endogenous NF subunits, that these soluble NF populations represent precursors for incorporation into the axonal cytoskeleton.

Herbicidins A and B, two new antibiotics with herbicidal activity II. Fermentation, isolation and physico-chemical characterization.

Herbicidins were produced in submerged fermentation by Streptomyces sagonomensis. Isolation of the antibiotics from the culture broth was performed by adsorption on resinous adsorbent followed by elution with aqueous acetone. Herbicidins A and B were separated from each other by counter-current distribution on a Ronor column or by silica gel chromatography. Physico-chemical characterization revealed that herbicidins are new antibiotics having an adenine nucleoside moiety in their structures.