All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells

While it is clear that a single hematopoietic stem cell?(HSC) is capable of giving rise to all other hematopoietic cell types, the differentiation paths beyond HSC remain controversial. Contradictory reports on?the lineage potential of progenitor populations have questioned their physiological contribution of progenitor populations to multilineage differentiation. Here, we established a lineage tracing mouse model that enabled direct assessment of differentiation pathways in?vivo. We provide definitive evidence that differentiation into all hematopoietic lineages, including megakaryocyte/erythroid cell types, involves Flk2-expressing non-self-renewing progenitors. A Flk2+ stage was used during steady-state hematopoiesis, after irradiation-induced stress and upon HSC transplantation. In contrast, HSC origin and maintenance do not include a Flk2+ stage. These data demonstrate that HSC specification and maintenance are Flk2 independent, and that hematopoietic lineage separation occurs downstream of Flk2 upregulation.     

Circulating gonadotrophins and follicular dynamics in anestrous ewes after treatment with estradiol-17beta.

Plasma FSH, LH, estradiol (E2) and progesterone (P4) profiles and patterns of follicular growth and regression by ultrasonography were determined after E2 treatment (1 microg/kg) in anestrous ewes. Fifteen ewes were treated with one (group I, n=7) or two (group II, n=4) i.m. injections of E2 with a 24h interval, or two oil injections with a 24h interval (group C, n=4). Blood samples for E2, P4, FSH and LH determinations were collected daily 4 days before the initiation of the treatment (day 0), when bleeding increased to every 2h starting 2h before treatment until 56h after the first injection and from then on every 6h until day 8, and twice per day till the end of the experiment (day 9). During the experimental period (days -4 to 9), transrectal ultrasonic examinations were carried out daily using a 7.5 MHz linear array probe. Number and size of follicles > or =3mm in diameter were recorded. No estrous was detected before, during or after treatment. LH and FSH surges were observed 10-18h after the first E2 injection. The second E2 injection stimulated another release of LH but no surges. E2 inhibited FSH levels before the surge and the second E2 injection induced a longer inhibition. No ovulation was detected by ultrasonography during the experimental period and P4 levels remained low (<0.7 nmol/l) before, during and after the treatment in all ewes. There was an effect of E2 treatment on the diameter of the largest follicle, a decrease could be observed 3 days after the first injection in both ewes of groups I and II. The E2-treated groups had a higher frequency of ewes showing wave emergence on day 3 (day 1.5+/-1,2.4+/-0.4 and 2.5+/-0.5 for control, groups I and II). LH and FSH surges were observed after E2 treatment, but were not able to provoke ovulation neither luteinization. In contrast, the treatment was associated with the regression of the largest follicle and with emergence of a new follicular wave on day 3.     

Divergence time of porcine reproductive and respiratory syndrome virus subtypes

Porcine reproductive and respiratory syndrome virus (PRRSV) recently emerged in domestic pigs of Western Europe and North America. Although time of emergence was identical on the two continents, genetic composition was markedly different with a clear geographical subtype structure, indicating that subtypes diverged in separate reservoirs prior to emergence. Genetic analyses have shown that the most recent common ancestor (MRCA) of Western European isolates existed around 1980 and that these originate from Eastern European pigs. These findings are challenged by a study of Hanada et al. who place the MRCA of all PRRSV isolates around 1980 and find that no significant subtype divergence occurred before emergence. Here, I discuss problems of information content, methodology, and biological plausibility associated with this study. Using alternative methodology, I reanalyze the existing data and conclude that the MRCA of all PRRSV isolates existed around 1880, 100 years before the date estimated by Hanada et al.     

Hysterographic appearance and uterine histology at different stages of the reproductive cycle and after progestagen treatment in the domestic cat

The aims of this study were to characterize the hysterographic and histological features of the uteri and to perform immunohistochemistry with proliferating cell nuclear antigen (PCNA) in the cat endometrium at various stages of the reproductive cycle and after treatment with exogenous progestagen. Seventy-four female domestic cats submitted for routine ovariohysterectomy were categorized into six groups: inactive (n=20), follicular (n=9), luteal (n=18), and postpartum (n=12) stages of the reproductive cycle; cats given medroxyprogesterone acetate for estrus prevention (MPA group) (n=12); and cats with uterine pathological lesions (n=3). Hysterography was performed and the relation of the uterine and luminal shape in the hysterogram with the stage of the reproductive cycle as well as with any pathological conditions of the uterus was evaluated. The uteri and ovaries were thereafter surgically removed and sectioned for histological examination. The PCNA was used to demonstrate the expression of endometrial epithelial cell growth. The hysterographic appearance was found to differ between the six groups of cats. A straight uterine cavity was characteristic for cats in the inactive stage, whereas a wavy uterine cavity was characteristic for cats in the follicular stage. In the luteal stage, the luminal cavity of the uteri differed in shape with increasing progesterone concentration from straight to irregular wavy or coiled. The coil shaped uterine lumen seen in the MPA treated and pathological groups was considered also to be an expression of a progestagenic effect. Waviness and coiling of the uterine lumen was related to a proliferation of the endometrial glands, whereas irregular filling defects were indicative of endometrial cystic changes. This study is the first to demonstrate the expression of PCNA in the cat endometrium although no differences were found between the six groups of cats. The hysterographic appearance was found to differ according to stage of the reproductive cycle and pathological conditions. Thus, a normative hysterogram is now available for diagnosing the reproductive stage and uterine changes in cats developing endometrial hyperplasia with and without cystic changes.     

Protein profiling of human pancreatic islets by two-dimensional gel electrophoresis and mass spectrometry

Completion of the human genome sequence has provided scientists with powerful resources with which to explore the molecular events associated with disease states such as diabetes. Understanding the relative levels of expression of gene products, especially of proteins, and their post-translational modifications will be critical. However, though the pancreatic islets play a key role in glucose homeostasis, global protein expression data in human are decidedly lacking. We here report the two-dimensional protein map and database of human pancreatic islets. A high level of reproducibility was obtained among the gels and a total of 744 protein spots were detected. We have successfully identified 130 spots corresponding to 66 different protein entries and generated a reference map of human islets. The functionally characterized proteins include enzymes, chaperones, cellular structural proteins, cellular defense proteins, signaling molecules, and transport proteins. A number of proteins identified in this study (e.g., annexin A2, elongation factor 1-alpha 2, histone H2B.a/g/k, heat shock protein 90 beta, heat shock 27 kDa protein, cyclophilin B, peroxiredoxin 4, cytokeratins 7, 18, and 19) have not been previously described in the database of mouse pancreatic islets. In addition, altered expression of several proteins, like GRP78, GRP94, PDI, calreticulin, annexin, cytokeratins, profilin, heat shock proteins, and ORP150 have been associated with the development of diabetes. The data presented in this study provides a first-draft reference map of the human islet proteome, that will pave the way for further proteome analysis of pancreatic islets in both healthy and diabetic individuals, generating insights into the pathophysiology of this condition.     

The YopD translocator of Yersinia pseudotuberculosis is a multifunctional protein comprised of discrete domains

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III translocon to microinject several anti-host Yop effectors into the cytosol of target eukaryotic cells. YopD has been implicated in several key steps during Yop effector translocation, including maintenance of yop regulatory control and pore formation in the target cell membrane through which effectors traverse. These functions are mediated, in part, by an interaction with the cognate chaperone, LcrH. To gain insight into the complex molecular mechanisms of YopD function, we performed a systematic mutagenesis study to search for discrete functional domains. We highlighted amino acids beyond the first three N-terminal residues that are dispensable for YopD secretion and confirmed that an interaction between YopD and LcrH is essential for maintenance of yop regulatory control. In addition, discrete domains within YopD that are essential for both pore formation and translocation of Yop effectors were identified. Significantly, other domains were found to be important for effector microinjection but not for pore formation. Therefore, YopD is clearly essential for several discrete steps during efficient Yop effector translocation. Recognition of this modular YopD domain structure provides important insights into the function of YopD.     

Deficiencies in gut NK cell number and function precede diabetes onset in BB rats

Defects in the intestinal immune system may contribute to the pathogenesis of autoimmune diseases. Intraepithelial lymphocytes represent a substantial fraction of gut-associated lymphocytes, but their function in mucosal immunity is unclear. A newly described population of NK cells that spontaneously secrete IL-4 and IFN-gamma is present in the intraepithelial lymphocyte compartment of the rat. We hypothesized that defects in the number or function of these cells would be present in rats susceptible to autoimmunity. We report that the number of NKR-P1A(+)CD3(-) intraepithelial NK (IENK) cells is deficient before onset of spontaneous autoimmune diabetes in diabetes-prone BB (BBDP) rats. The absolute number of recoverable IENK cells was only approximately 8% of that observed in WF rats. Bone marrow transplantation from histocompatible WF donors reversed the IENK cell deficiency (and prevented diabetes) in these animals, suggesting a hemopoietic origin for their IENK cell defect. Analysis of diabetes-resistant BB rats, which develop autoimmune diabetes only after perturbation of the immune system, revealed IENK cell numbers intermediate between that of BBDP and WF rats. IENK cells were selectively depleted during treatment to induce diabetes. Prediabetic BBDP and diabetes-resistant BB animals also exhibited defective IENK cell function, including decreased NK cell cytotoxicity and reduced secretion of IL-4 and IFN-gamma. IENK functional defects were also observed in LEW and BN rats, which are susceptible to induced autoimmunity, but not in WF, DA, or F344 rats, which are resistant. Defects in IENK cell number and function may contribute to the pathogenesis of autoimmune diseases including type 1 diabetes.     

Tetratricopeptide repeats are essential for PcrH chaperone function in Pseudomonas aeruginosa type III secretion

The type III secretion system (T3SS) is a specialized apparatus evolved by Gram-negative bacteria to deliver effector proteins into host cells, thus facilitating the establishment of an infection. Effector translocation across the target cell plasma membrane is believed to occur via pores formed by at least two secreted translocator proteins, the functions of which are dependent upon customized class II T3SS chaperones. Recently, three internal tetratricopeptide repeats (TPRs) were identified in this class of chaperones. Here, defined mutagenesis of the class II chaperone PcrH of Pseudomonas aeruginosa revealed these TPRs to be essential for chaperone activity towards the translocator proteins PopB and PopD and subsequently for the translocation of exoenzymes into host cells.     

Structural characterization of a K-antigen capsular polysaccharide essential for normal symbiotic infection in Rhizobium sp. NGR234: deletion of the rkpMNO locus prevents synthesis of 5,7-diacetamido-3,5,7,9-tetradeoxy-non-2-ulosonic acid

Many early molecular events in symbiotic infection have been documented, although factors enabling Rhizobium to progress within the plant-derived infection thread and ultimately survive within the intracellular symbiosome compartment as mature nitrogen-fixing bacteroids are poorly understood. Rhizobial surface polysaccharides (SPS), including the capsular polysaccharides (K-antigens), exist in close proximity to plant-derived membranes throughout the infection process. SPSs are essential for bacterial survival, adaptation, and as potential determinants of nodulation and/or host specificity. Relatively few studies have examined the role of K-antigens in these events. However, we constructed a mutant that lacks genes essential for the production of the K-antigen strain-specific sugar precursor, pseudaminic acid, in the broad host range Rhizobium sp. NGR234. The complete structure of the K-antigen of strain NGR234 was established, and it consists of disaccharide repeating units of glucuronic and pseudaminic acid having the structure -->4)-beta-d-glucuronic acid-(1-->4)-beta-5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid-(2-->. Deletion of three genes located in the rkp-3 gene cluster, rkpM, rkpN, and part of rkpO, abolished pseudaminic acid synthesis, yielding a mutant in which the strain-specific K-antigen was totally absent: other surface glycoconjugates, including the lipopolysaccharides, exopolysaccharides, and flagellin glycoprotein appeared unaffected. The NGRDeltarkpMNO mutant was symbiotically defective, showing reduced nodulation efficiency on several legumes. K-antigen production was found to decline after rhizobia were exposed to plant flavonoids, and the decrease coincided with induction of a symbiotically active (bacteroid-specific) rhamnan-LPS, suggesting an exchange of SPS occurs during bacterial differentiation in the developing nodule.