Short communication: Effects of a commercial triple-strain Bacillus-based probiotic on cecal colonization with Salmonella Enteritidis in commercial layer pullets.

A commercial triple-strain Bacillus-based probiotic was tested to determine its effect on the colonization of the ceca by Salmonella Enteritidis (SE) in commercial layer pullets. Two treatments were tested, each with containing 128 day-of-hatch LSL layer chicks. On top of a standard diet: 1) no supplement (Control, CON), and 2) Probiotic (GalliPro® Fit, 500 g/MT, 1.6 × 106 CFU/g of finished feed, PRO). Environmental swabs were collected from each treatment group and tested to ensure freedom from SE prior to challenge. At 21 days of age, the SE challenge strain was administered orally at a dose of 3.3 × 108 CFU/bird. Pullets from each treatment group (n=32) were euthanized at 6-, 10-, 14-, and 18-days post infection (dpi). Contents from the ceca were aseptically collected and assessed for presence and abundance of SE. No differences in the prevalence of SE positive ceca following oral inoculation (P>0.05) were observed between treatment groups at 6-, 10-, 14-, or 18-dpi. Counts of SE in the ceca of the PRO group were not significantly different (P>0.05) from those of CON at 6- or 10-dpi. However, significantly lower counts of SE in the ceca of the PRO group were observed at 14-dpi (P<0.05) and 18-dpi (P<0.05) compared with CON. SE counts were 1.24 and 1.34 logs lower than CON at 14- and 18-dpi, respectively. In conclusion, supplementation of the triple-strain Bacillus-based probiotic resulted in lower cecal counts of SE compared to those that did not receive an effective probiotic, thereby reducing the risk of foodborne pathogens prior to harvest through sustainable, natural methods.

Loss of the Parkinson's disease-linked gene DJ-1 perturbs mitochondrial dynamics.

Growing evidence highlights a role for mitochondrial dysfunction and oxidative stress as underlying contributors to Parkinson's disease (PD) pathogenesis. DJ-1 (PARK7) is a recently identified recessive familial PD gene. Its loss leads to increased susceptibility of neurons to oxidative stress and death. However, its mechanism of action is not fully understood. Presently, we report that DJ-1 deficiency in cell lines, cultured neurons, mouse brain and lymphoblast cells derived from DJ-1 patients display aberrant mitochondrial morphology. We also show that these DJ-1-dependent mitochondrial defects contribute to oxidative stress-induced sensitivity to cell death since reversal of this fragmented mitochondrial phenotype abrogates neuronal cell death. Reactive oxygen species (ROS) appear to play a critical role in the observed defects, as ROS scavengers rescue the phenotype and mitochondria isolated from DJ-1 deficient animals produce more ROS compared with control. Importantly, the aberrant mitochondrial phenotype can be rescued by the expression of Pink1 and Parkin, two PD-linked genes involved in regulating mitochondrial dynamics and quality control. Finally, we show that DJ-1 deficiency leads to altered autophagy in murine and human cells. Our findings define a mechanism by which the DJ-1-dependent mitochondrial defects contribute to the increased sensitivity to oxidative stress-induced cell death that has been previously reported.

Emergence of immunoglobulin variants following treatment of a B cell leukemia with an immunotoxin composed of antiidiotypic antibody and saporin.

The potency and specificity of immunotoxins consisting of monoclonal antiidiotype conjugated to the ribosome-inactivating protein, saporin, have been evaluated in the treatment of guinea pig L2C B lymphocytic leukemia. The immunotoxins were therapeutically much more effective than their parent antibodies. Their specificity reflected that of their antiidiotype component. Although the leukemia emerged eventually in most animals treated with these conjugates, most of the cells showed altered Ig expression, which rendered them resistant to the therapy. Commonly, the emerging cells had lost mu heavy chain production, leaving them negative for intracellular, surface, and secreted IgM, but still positive for lambda light chain production. In addition, a minor group of L2C variants was identified in a protocol designed to detect mutants at very low frequency: here the cells were exposed in vitro to immunotoxin and, while still viable as judged by dye-exclusion, inoculated in large numbers into animals. In tumor that emerged under these circumstances, the majority of cells were again immunoglobulin-negative; however a minority exhibited IgM with an altered idiotype (Idiotope-loss variants), rendering them unreactive with immunotoxin. Immunotherapy with unmodified anti-Id antibody alone does not reveal these variants, and we suggest it is the increased selective force exerted by the highly potent immunotoxins that allow these minor nonreactive populations to emerge.

The human mitochondrial import receptor, hTom20p, prevents a cryptic matrix targeting sequence from gaining access to the protein translocation machinery.

Yeast Mas70p and NADH cytochrome b5 reductase are bitopic integral proteins of the mitochondrial outer membrane and are inserted into the lipid-bilayer in an Nin-Ccyto orientation via an NH2-terminal signal-anchor sequence. The signal anchor of both proteins is comprised of a short, positively charged domain followed by the predicted transmembrane segment. The positively charged domain is capable of functioning independently as a matrix-targeting signal in yeast mitochondria in vitro but does not support import into mammalian mitochondria (rat or human). Rather, this domain represents a cryptic signal that can direct import into mammalian mitochondria only if proximal components of the outer membrane import machinery are removed. This can be accomplished either by treating the surface of the intact mitochondria with trypsin or by generating mitoplasts. The import receptor Tom20p (Mas20p/MOM19) is responsible for excluding the cryptic matrix-targeting signal from mammalian mitochondria since replacement of yeast Tom20p with the human receptor confers this property to the yeast organelle while at the same time maintaining import of other proteins. In addition to contributing to positive recognition of precursor proteins, therefore, the results suggest that hTom20p may also have the ability to screen potential matrix-targeting sequences and exclude certain proteins that would otherwise be recognized and imported by distal components of the outer and inner membrane protein-translocation machinery. These findings also indicate, however, that cryptic signals, if they exist within otherwise native precursor proteins, may remain topogenically silent until the precursor successfully clears hTom20p, at which time the activity of the cryptic signal is manifested and can contribute to subsequent translocation and sorting of the polypeptide.

A signal-anchor sequence selective for the mitochondrial outer membrane.

pOMD29 is a hybrid protein containing the NH2-terminal topogenic sequence of a bitopic, integral protein of the outer mitochondrial membrane in yeast, OMM70, fused to dihydrofolate reductase. The topogenic sequence consists of two structural domains: an NH2-terminal basic region (amino acids 1-10) and an apolar region which is the predicted transmembrane segment (amino acids 11-29). The transmembrane segment alone was capable of targeting and inserting the hybrid protein into the outer membrane of intact mitochondria from rat heart in vitro. The presence of amino acids 1-10 enhanced the rate of import, and this increased rate depended, in part, on the basic amino acids located at positions 2, 7, and 9. Deletion of a large portion of the transmembrane segment (amino acids 16-29) resulted in a protein that exhibited negligible import in vitro. Insertion of pOMD29 into the outer membrane was not competed by import of excess precursor protein destined for the mitochondrial matrix, indicating that the two proteins may have different rate-limiting steps during import. We propose that the structural domains within amino acids 1-29 of pOMD29 cooperate to form a signal-anchor sequence, the characteristics of which suggest a model for proper sorting to the mitochondrial outer membrane.

Tumorigenicity in human melanoma cell lines controlled by introduction of human chromosome 6.

Chromosome banding analysis of human malignant melanoma has documented the nonrandom alteration of chromosome 6. To determine the relevance of chromosome 6 abnormalities in melanoma, a normal chromosome 6 was directly introduced into melanoma cell lines. The resulting (+6) microcell hybrids were significantly altered in their phenotypic properties in culture and lost their ability to form tumors in nude mice. The loss of the chromosome 6 from melanoma microcell hybrids resulted in the reversion to tumorigenicity of these cells in mice. The introduction of the selectable marker (psv2neo) alone into melanoma cell lines had no effect on tumorigenicity. These results support the idea that one or more genes on chromosome 6 may control the malignant expression of human melanoma.

Long-range interactions at the HO promoter.

The SWI5 gene encodes a zinc finger DNA-binding protein required for the transcriptional activation of the yeast HO gene. There are two Swi5p binding sites in the HO promoter, site A at -1800 and site B at -1300. Swi5p binding at site B has been investigated in some detail, and we have shown that Swi5p binds site B in a mutually cooperative fashion with Pho2p, a homeodomain protein. In this report, we demonstrate that Swi5p and Pho2p bind cooperatively to both sites A and B but that there are differences in binding to these two promoter sites. It has been shown previously that point mutations in either Swi5p binding site only modestly reduce HO expression in a PHO2 strain. We show that these mutant promoters are completely inactive in a pho2 mutant. We have created stronger point mutations at the two Swi5p binding sites within the HO promoter, and we show that the two binding sites, separated by 500 bp, are both absolutely required for HO expression, independent of PHO2. These results create an apparent dilemma, as the strong mutations at the Swi5p binding sites show that both binding sites are required for HO expression, but the earlier binding site mutations allow Swi5p to activate HO, but only in the presence of Pho2p. To explain these results, a model is proposed in which physical interaction between Swi5p proteins bound to these two sites separated by 500 bp is required for activation of the HO promoter. Experimental evidence is presented that supports the model. In addition, through deletion analysis we have identified a region near the amino terminus of Swi5p that is required for PHO2-independent activation of HO, suggesting that this region mediates the long-range interactions between Swi5p molecules bound at the distant sites.

Degradation of the transcription factor Gcn4 requires the kinase Pho85 and the SCF(CDC4) ubiquitin-ligase complex.

Gcn4, a yeast transcriptional activator that promotes the expression of amino acid and purine biosynthesis genes, is rapidly degraded in rich medium. Here we report that SCF(CDC4), a recently characterized protein complex that acts in conjunction with the ubiquitin-conjugating enzyme Cdc34 to degrade cell cycle regulators, is also necessary for the degradation of the transcription factor Gcn4. Degradation of Gcn4 occurs throughout the cell cycle, whereas degradation of the known cell cycle substrates of Cdc34/SCF(CDC4) is cell cycle regulated. Gcn4 ubiquitination and degradation are regulated by starvation for amino acids, whereas the degradation of the cell cycle substrates of Cdc34/SCF(CDC4) is unaffected by starvation. We further show that unlike the cell cycle substrates of Cdc34/SCF(CDC4), which require phosphorylation by the kinase Cdc28, Gcn4 degradation requires the kinase Pho85. We identify the critical target site of Pho85 on Gcn4; a mutation of this site stabilizes the protein. A specific Pho85-Pcl complex that is able to phosphorylate Gcn4 on that site is inactive under conditions under which Gcn4 is stable. Thus, Cdc34/SCF(CDC4) activity is constitutive, and regulation of the stability of its various substrates occurs at the level of their phosphorylation.

In vivo and in vitro examination of the functional significances of novel lamin gene mutations in heart failure patients.

CONTEXT: Lamin A/C (LMNA) gene variations have been reported in more than one third of genotyped families with dilated cardiomyopathy (DCM). However, the relationship between LMNA mutation and the development of DCM is poorly understood. METHODS AND RESULTS: We found that end stage DCM patients carrying LMNA mutations displayed either dramatic ultrastructural changes of the cardiomyocyte nucleus (D192G) or nonspecific changes (R541S). Overexpression of the D192G lamin C dramatically increased the size of intranuclear speckles and reduced their number. This phenotype was only partially reversed by coexpression of the D192G and wild type lamin C. Moreover, the D192G mutation precludes insertion of lamin C into the nuclear envelope when co-transfected with the D192G lamin A. By contrast, the R541S phenotype was entirely reversed by coexpression of the R541S and wild type lamin C. As lamin speckle size is known to be correlated with regulation of transcription, we assessed the SUMO1 distribution pattern in the presence of mutated lamin C and showed that D192G lamin C expression totally disrupts the SUMO1 pattern. CONCLUSION: Our in vivo and in vitro results question the relationship of causality between LMNA mutations and the development of heart failure in some DCM patients and therefore, the reliability of genetic counselling. However, LMNA mutations producing speckles result not only in nuclear envelope structural damage, but may also lead to the dysregulation of cellular functions controlled by sumoylation, such as transcription, chromosome organisation, and nuclear trafficking.

Insertion of an uncharged polypeptide into the mitochondrial inner membrane does not require a trans-bilayer electrochemical potential: effects of positive charges.

Mitochondria with a ruptured outer membrane exhibited impaired import into this membrane of an outer membrane fusion protein containing the signal-anchor sequence of Mas70p. However, the Mas70p signal-anchor efficiently targeted and inserted the protein directly into exposed regions of the inner membrane. Import into the inner membrane was dependent on delta psi and this dependence was due to the presence of the positively-charged amino acids located at positions 2, 7, and 9 of the signal-anchor. In contrast to wild-type signal-anchor, mutants lacking the positively-charged residues mediated import into the inner membrane in both the presence and absence of delta psi. The results suggest two conclusions: (1) delta psi-dependent import of the signal-anchor sequence was due exclusively to an effect of delta psi on the positively-charged domain of the signal-anchor, rather than to an effect of delta psi on a property of the inner membrane import machinery; (2) in the absence of delta psi, the positively-charged domain of the signal-anchor prevented the otherwise import-competent signal-anchor from inserting into the membrane. This suggests that the positively-charged domain leads import across the inner membrane, and that delta psi is required to vectorially clear this domain in order to allow the distal region of the signal-anchor to enter the translocation pathway. The implications of these findings on the mechanism of import into the mitochondrial inner membrane and matrix are discussed.

The human steroidogenic acute regulatory (StAR) gene is expressed in the urogenital system and encodes a mitochondrial polypeptide.

The first enzymatic step in the biosynthesis of steroid hormones occurs in the mitochondrial inner membrane and is dependent on the mobilization of cholesterol from cellular stores. We report on the isolation of a human cDNA which encodes a mitochondrial protein called steroidogenic acute regulatory (StAR) protein, implicated in transport of cholesterol into mitochondria. Nucleotide and predicted amino acid sequence analyses indicate that the human and murine polypeptides are highly conserved, sharing 87% identity with an overall homology of 92%. Analysis of the distribution of StAR mRNA transcripts in human tissues by Northern blotting reveals several mRNA species, the most abundant of which is a 1.8 kb mRNA transcript present in testes, ovaries and kidneys. Using in vitro translated protein, we demonstrate that the StAR gene product can be efficiently imported into exogenously added mitochondria.

Differentiation of alcoholics. Childhood history of minimal brain dysfunction, family history, and drinking pattern.

Alcoholics were differentiated into two subgroups on the basis of drinking patterns and subjective response to alcohol. Severe drinkers (primary alcoholics) retrospectively reported more symptoms of childhood minimal brain dysfunction than less severe drinkers (secondary alcoholics), psychiatric patients, and normals. The alcoholics as a group reported a greater incidence of familial alcohol abuse than the psychiatric subjects, but a difference on this factor was not observed between the primary and secondary subgroups. In terms of clinical status, the primary alcoholics presented Minnesota Multiphasic Personality Inventory profile more indicative of normality than the other groups, but scored significantly higher on the MacAndrew Alcoholism Scale. These findings are discussed in light of further delineating a specific subtype of alcoholism that may have a genetic-constitutional relationship with other pathological disorders.

Double immunohistochemistry enhances detection of lymphatic and venous invasion in early-stage colorectal cancer.

Lymphatic invasion (LI) and venous invasion (VI) are regarded as important risk factors of nodal disease in early-stage colorectal cancer (CRC) but with variable reporting and poor distinction of these parameters in previous studies. This study examines the application of a double immunohistochemistry (D-IHC) method to help detect and distinguish LI and VI, in comparison with haematoxylin and eosin (H&E) staining, in a clinical series of cases of stage pT1 CRC. The aims were to demonstrate feasibility of this methodology in routine practice and compare rates of LI and VI reporting with and without D-IHC application. D-IHC utilising CAM5.2 with the endothelial marker CD34 and with the specific lymphatic endothelial marker D2-40 was performed on parallel sections from single representative paraffin tissue blocks in 28 cases of stage pT1 CRC from routine clinical practice. D-IHC significantly increased rates of both LI and VI reporting, from 14.3 to 35.7 % and from 14.3 to 28.6 %, respectively. The D-IHC methodology described is technically feasible in routine practice and potentially offers a more sensitive and robust assay for detection and distinction of LI and VI in early CRC pathology reporting. The reproducibility and clinical significance of enhanced LI and VI detection by this method and the relative importance of LI and VI in this clinical setting require further study.

Development of core competencies in clinical nutrition.

A nationwide survey of core competencies of nutrition knowledge and skills that primary care physicians should know and/or be able to do has been completed. Nutrition competencies were synthesized from a review of clinical nutrition practices as reported in the medical literature and by medical school faculty group discussions. A nutrition competency questionnaire was sent to 445 practicing physicians and to 752 department chairpersons in every US medical school in the disciplines of family practice, medicine, obstetrics and gynecology, pediatrics, psychiatry, and surgery. The overall response rate for practitioners and faculty combined was 46.03%. Of the 55 nutrition competencies, 28 items received very strong support (greater than 90% of respondents marked agree or completely agree). Twenty-two competencies received strong support (80 to 89% of respondents marked agree or completely agree), and five items received mixed support (40 to 79% of respondents marked agree or completely agree). Statistical comparisons of the item responses between practitioners and faculty were significantly different (p less than 0.05) on 19 (35%) of the items. It is anticipated that the 50 nutrition competencies that have received strong or very strong agreement among the 551 physicians representing primary care disciplines across the US will serve as guidelines for continued development of medical school curriculum and continuing medical education in clinical nutrition.

Possible relationship between chromosome alterations and in vitro cellular radiosensitivity of human malignant melanoma.

In the present study, in vitro radiation survival analysis was performed on 8 human malignant melanoma cell lines with defined karyotypes. Exponentially growing cells were irradiated to doses of 0 to 8 Gy and examined for soft agar clonogenicity. Two groups emerged from this analysis: 4 cell lines with a small shoulder (extrapolation number, n less than 2) and 4 cell lines with a large shoulder (n greater than 2). Of possible significance, 4/4 cell lines with extrapolation numbers greater than 2 demonstrated clonal structural abnormalities of chromosome 7. In contrast, 3/4 cell lines with extrapolation numbers less than 2 had no structural abnormalities of chromosome 7. These very preliminary results suggest that structural alterations of chromosome 7 may be associated with melanoma tumors with high extrapolation numbers.

Attention deficit disorder and pathological gambling.

To examine the possibility that pathological gambling is related to the deficits in impulse control associated with attention deficit disorder, 14 pathological gamblers and 16 controls were administered questionnaires concerning their childhood behaviors. These self-reports indicated a strong correlation between pathological gambling and childhood behaviors related to attention deficit disorder.

Cytomegalovirus infection of the cervix: morphological observations in five cases of a possibly under-recognised condition.

AIMS: Histologically diagnosed cytomegalovirus (CMV) infection of the cervix is rare and the associated morphological features are not well described. This study describes histopathological findings in five biopsies from four patients with CMV cervicitis. METHODS: CMV inclusions were identified in five cervical biopsies from four patients in a single institution over eight months. The clinical notes were reviewed, the morphological features documented, and immunohistochemical staining for CMV performed. CMV immunohistochemical staining was also performed on 30 consecutive cervical biopsies in which inclusions were not seen histologically. RESULTS: None of the patients was immunocompromised but one was postnatal. Numbers of CMV inclusions ranged from occasional to abundant and they were located mainly in endocervical glandular epithelial cells but also in endothelial and mesenchymal cells. Inclusions were not seen in squamous cells. Inclusions were eosinophilic and were intracytoplasmic rather than intranuclear. They were positive immunohistochemically for CMV. Associated morphological features included fibrin thrombi within small blood vessels (three cases), dense active inflammatory infiltrates (five cases), lymphoid follicles (two cases), vacuolation of glandular epithelial cells (two cases), and reactive changes in glandular epithelial cells (two cases). CMV inclusions were not identified in the 30 additional cases that underwent immunohistochemical staining. CONCLUSIONS: CMV infection of the cervix may be more common than is thought. Patients are usually immunocompetent and require no treatment. Morphological features such as a dense inflammatory cell infiltrate with lymphoid follicles, and especially fibrin thrombi within small vessels, should alert the pathologist to look closely for the pathognomonic CMV inclusion bodies.

Immunohistochemical staining of plastic embedded bone marrow trephine biopsy specimens after microwave heating.

AIMS: To investigate (1) whether adequate immunohistochemical staining can be achieved on sections cut from plastic embedded bone marrow trephine biopsy specimens after microwave heating in citrate buffer; and (2) whether this immunohistochemical staining is comparable with that achieved on routine sections cut from paraffin wax embedded trephine biopsy specimens after decalcification procedures. METHODS: Sixty five consecutive bone marrow trephine biopsy specimens of more than 1 cm in length were divided transversely into two equal parts. One part was processed in paraffin wax followed by decalcification. The other part was embedded in the epoxyresin Polarbed 812 followed by the cutting of 1 micron sections. Both parts underwent immunohistochemical staining by an identical panel of antibodies. With Polarbed 812 plastic embedded sections, microwave heating in citrate buffer was undertaken before the application of antisera. RESULTS: On sections cut from plastic embedded material, immunohistochemical staining was generally satisfactory, easy to interpret and comparable with that achieved with paraffin wax embedded material. Exceptions were antibodies to neutrophil elastase and CD61 where immunostaining was consistently negative on plastic embedded sections. Immunohistochemical staining for CD20 was consistently more reliable on plastic embedded sections. CONCLUSIONS: The results provide evidence that, with few exceptions, satisfactory immunohistochemical staining is possible on plastic embedded bone marrow trephine biopsy specimens after microwave heating in citrate buffer. This, combined with the advantage of superior cellular morphology with semi-thin (1 micron) sections of plastic embedded material, make such embedding procedures the preferred method for the processing of bone marrow trephine biopsy specimens.

Adrenal hemorrhage: a complication of anticoagulant therapy--a case history.

A seventy-five year-old woman developed adrenal hemorrhage and acute adrenal insufficiency while receiving anticoagulant therapy. Abdominal CT scan was consistent with bilateral adrenal hemorrhage and was an important contribution to diagnosis and therapy. Acute adrenal hemorrhage should be suspected in patients, especially the elderly, who have recently begun anticoagulant therapy and develop upper abdominal pain followed by decreased sensorium, high fever, hypotension, and hyponatremia. Any consideration of the diagnosis of sepsis with shock in a recently anticoagulated elderly hospital patient should suggest the possibility of acute adrenal hemorrhage. Abdominal CT scan and a cosyntropin stimulation test should be performed to confirm the diagnosis. Failure of diagnosis has generally been associated with death in most patients, whereas prognosis in patients treated with corticosteroids is excellent.

Monoclonal antibodies raised against the idiotype of the murine B cell lymphoma, BCL1 act primarily with heavy chain determinants.

Anti-idiotypic antibodies can be used as probes to distinguish neoplastic cells from their normal counterparts. In addition they have been used in the passive therapy of B cell tumors. In this report we describe a panel of 7 rat monoclonal antibodies raised against idiotypic determinants carried by the IgM molecule of the BCL1 lymphoma. The majority (6/7) of these antibodies recognize private idiotypic determinants that are carried on the isolated mu heavy chain of the molecule, and do not require the lambda chain for reactivity. This is unusual for antibodies raised against the idiotype of the whole immunoglobulin molecule, which normally require both chains for reactivity. The antibodies do not, however, bind peptides corresponding to the complementarity determining regions of the mu heavy chain of BCL1. The antibodies perform well in complement mediated cytotoxicity, and, in at least one case, are effective in the passive immunotherapy of BCL1 lymphoma.