Characterization of the Particles of Purified κ-Casein: Trypsin as a Probe of Surface-Accessible Residues

κ-Casein as purified from bovine milk exhibits a rather unique disulfide bonding pattern as revealed by SDS–PAGE. The disulfide-bonded caseins present range from dimer to octamer and above and preparations contain about 10% monomer. All of these heterogeneous polymers, however, self-associate into nearly spherical particles with an average diameter of 13 nm at pH 8.0, as revealed by negatively stained transmission electron micrographs and dynamic light scattering. The weight-average molecular weight of the aggregates at pH 8.0, as judged by analytical ultracentrifugation, is 648,000. Trypsin digestion at pH 8.0 was used to probe the surface groups of the κ-casein A polymers. The reaction with trypsin was rapid and the peptides liberated were identified by separation with reverse-phase HPLC, amino acid analysis, and protein sequencing. The most rapidly released peptides (t _1/2 < 30 sec) were from cleavage at Arg 97 and Lys residues 111 and 112. These results suggest a surface orientation for these residues, and the data are in accord with earlier proposed 3D predictive models for κ-casein. It is speculated that Arg 97, together with adjacent His residues (98 and 100) and Lys residues 111 and 112, form two positively charged clusters on the surface of the otherwise negatively charged casein. These clusters bracket the neutral chymosin cleavage site (whose hydrolysis triggers a well-known digestive process) and so these clusters may facilitate docking of the substrate caseins with chymosin.     

Part II: beneficial effects of the peroxynitrite decomposition catalyst FP15 in murine models of arthritis and colitis

BACKGROUND: Peroxynitrite is a reactive oxidant species produced from nitric oxide and superoxide, which has been indirectly implicated in the pathogenesis of many inflammatory conditions including arthritis and colitis. Here, using a novel peroxynitrite decomposition catalyst, FP15, we directly investigate the role of peroxynitrite in the pathogenesis of arthritis and colitis in rodent models. METHODS: Arthritis was induced in mice by intradermal collagen injection; incidence and severity of arthritis was monitored using a macroscopic scoring system. At the end of the experiment paws were taken for determination of neutrophil infiltration (myeloperoxidase [MPO] activity), oxidative stress (malondialdehyde [MDA] level), and cytokine/chemokine levels. Colitis was induced in mice by 5% dextran sodium sulfate (DSS) in their drinking water. Colitis symptoms were assessed 10 days later, the parameters determined included body weight, rectal bleeding, colon length, colonic MPO and MDA levels, and colon histologic damage. RESULTS: Treatment with FP15 significantly reduced the inflammation and oxidative stress in arthritis and colitis. FP15 reduced both the incidence and severity of arthritis in mice and this was associated with reduced paw MPO and MDA levels. Similarly, in colitis, FP15 reduced colon damage, and this was associated with reduced colon neutrophil infiltration and oxidative stress. CONCLUSIONS:The protective effect of FP15 suggests that peroxynitrite plays a significant pathogenetic role in arthritis and colitis in the currently employed rodent models. Further work is needed to determine whether neutralization of peroxynitrite also represents a promising strategy to treat human inflammatory diseases such as arthritis and colitis.     

Influence of prgH on the Persistence of Ingested Salmonella enterica in the Leafhopper Macrosteles quadrilineatus

Phytophagous insects can encounter Salmonella enterica on contaminated plant surfaces and transmit externally adhered and internalized bacteria on and among leaves. Excretion of ingested S. enterica by the leafhopper Macrosteles quadrilineatus has been previously reported; however, the sites of persistence of ingested bacteria remain undetermined. Fluorescence microscopy revealed the presence and persistence of S. enterica in various organs of M. quadrilineatus fed an inoculated diet for 12 h and then moved to two consecutive noninoculated diets for a total of 48 h. Ingested S. enterica was predominantly observed in the filter chamber, midgut, and Malpighian tubules of M. quadrilineatus dissected immediately after acquisition and at 24- and 48-h post-acquisition access periods (post-AAPs). Additionally, we examined the potential roles of the Salmonella pathogenicity island 1 (SPI-1) and SPI-2 type III secretion systems (T3SSs) in the persistence and excretion of ingested S. enterica. In competition assays, a prgH mutant lacking a functional SPI-1 T3SS was recovered at significantly lower levels than the WT in insect homogenates at 24 h post-AAP, and complementation with prgH restored S. enterica persistence in M. quadrilineatus. Moreover, expression of prgH inside M. quadrilineatus was observed up to 48 post-AAP. No differences were observed between the WT and an ssaK mutant lacking a functional SPI-2 T3SS in insect homogenates or between the WT and either mutant in insect excretions. This study provides novel insight into the presence and persistence of S. enterica inside M. quadrilineatus and demonstrates that the SPI-1 T3SS influences the persistence of the pathogen in the gut of a potential vector.     

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.     

The What,Why and How of Primate Taxonomy

Taxonomy has a well-defined role, which is much more than simply stamp-collecting and pigeon-holing. Species are the units of classification, biogeography and conservation; as such they must be defined as objectively as possible. The biological species concept, still widely used in biology, though predominantly by non-taxonomists and all too often misunderstood, is a process-based concept, which offers no criterion for the classification of allopatric populations beyond inference and hypothesis. The phylogenetic species concept—a pattern-based concept—is as nearly objective as we are likely to get. Amount of difference is not a criterion for recognizing species. It is not possible to insist on monophyly at the specific level, but it is mandatory for the higher categories (genus, family, etc.). The rank we assign to a given supraspecific category should be determined by its time depth.     

Kinship and sociality in coastal river otters: are they related?

Previous studies of coastal river otters (Lontra canadensis)in Prince William Sound, Alaska, USA, documented atypical socialorganization for mammals. Social groups were composed largelyof males, but some males remained solitary year-round and mostfemales were asocial. Because, in carnivores, groups are usuallycomposed of highly related individuals but group living alsoprovides advantages unrelated to kinship, we concurrently evaluatedthe role of relatedness and ecological benefits in socialityamong coastal river otters. By using DNA microsatellite analysisand radiotelemetry, we were able to reject the hypothesis thatsocial groups of otters were kin based. In addition, we foundno indication of kin avoidance, as would be expected from lowdispersal and high local competition. Sociality conferred noreproductive benefits or costs to otters; number of offspringand number of relatives in the population did not differ betweensocial and solitary animals. Solitary males were not older orlarger than were social males, and there was no relation betweenmale size and number of offspring, indicating that sexual selectiondid not mask a potential relation between sociality and reproductivesuccess. Among coastal river otters in this region, socialitycould be explained by the benefits obtained from cooperativeforaging on high-quality schooling pelagic fishes. Such benefitsdid not require association with kin, resulting in no selectionpressure for kin-based groups. The prediction that the degreeof sociality in the population will fluctuate relative to theabundance of schooling pelagic fishes merits further investigation.     

Seasonal population dynamics of Draeculacephala minerva (Hemiptera: Cicadellidae) and transmission of Xylella fastidiosa

The grass sharpshooter, Draeculacephala minerva Ball (Hemiptera: Cicadellidae), is a very common and often abundant grass-feeding leafhopper in California. Its population dynamics and ability to transmit Xylella fastidiosa were monitored over a 2-yr period in California's San Joaquin Valley. Collections of individuals from natural populations in irrigated pastures and alfalfa, Medicago savita L. fields adjacent to X. fastidiosa-infected almond (Prunus spp.) orchards indicated the occurrence of three discrete generations per year that peaked during the summer. Population densities varied significantly among experimental field survey sites. Insects captured on intercepting mesh traps, yellow sticky cards, and UV-light traps indicated local movement of these insects into and surrounding X. fastidiosa-infected, almond orchards. Local movement and seasonal transmission of X. fastidiosa from infected almonds to Catharanthus roseus (L.) G. Don indicated that this insect may be partly responsible for the slow spread of almond leaf scorch now recently observed in California's San Joaquin Valley.     

Lineage-restricted neural precursors can be isolated from both the mouse neural tube and cultured ES cells.

We have previously identified multipotent neuroepithelial (NEP) stem cells and lineage-restricted, self-renewing precursor cells termed NRPs (neuron-restricted precursors) and GRPs (glial-restricted precursors) present in the developing rat spinal cord (A. Kalyani, K. Hobson, and M. S. Rao, 1997, Dev. Biol. 186, 202-223; M. S. Rao and M. Mayer-Proschel, 1997, Dev. Biol. 188, 48-63; M. Mayer-Proschel, A. J. Kalyani, T. Mujtaba, and M. S. Rao, 1997, Neuron 19, 773-785). We now show that cells identical to rat NEPs, NRPs, and GRPs are present in mouse neural tubes and that immunoselection against cell surface markers E-NCAM and A2B5 can be used to isolate NRPs and GRPs, respectively. Restricted precursors similar to NRPs and GRPs can also be isolated from mouse embryonic stem cells (ES cells). ES cell-derived NRPs are E-NCAM immunoreactive, undergo self-renewal in defined medium, and differentiate into multiple neuronal phenotypes in mass culture. ES cells also generate A2B5-immunoreactive cells that are similar to E9 NEP-cell-derived GRPs and can differentiate into oligodendrocytes and astrocytes. Thus, lineage restricted precursors can be generated in vitro from cultured ES cells and these restricted precursors resemble those derived from mouse neural tubes. These results demonstrate the utility of using ES cells as a source of late embryonic precursor cells.     

Human beta-casein. Amino acid sequence and identification of phosphorylation sites

The primary structure of human beta-casein has been determined by automated Edman degradation of the intact protein and of peptides derived therefrom by hydrolysis with trypsin and by chemical cleavage with cyanogen bromide. For each form of this multiphosphorylated protein (0-5 P/molecule), phosphorylated sites at specific seryl and threonyl residues have been identified. These are located near the amino terminus, within the first 10 residues of this 212-amino acid molecule. Sequence comparison of human beta-casein with the bovine and ovine proteins reveals 50% identity and a 10-residue shifted alignment relationship. Locations of prolyl and charged residues are generally conserved for the three homologues. The sequence data indicate the existence of genetic polymorphism involving uncharged residues in human beta-casein.