Test of genetic heterogeneity of cleft lip with or without cleft palate as related to race and severity

The question of possible heterogeneity among population groups and phenotypic groups on the role of major gene in the etiology of cleft lip with or without cleft palate [CL(P)] was examined using the uniformly collected data in Hawaii. Complex segregation analysis was used to analyze patterns of family resemblance under the mixed model incorporating the effects of major gene and multifactorial inheritance. Analysis of the entire data showed superior fit of the mixed model including the effects of both major gene and multifactorial inheritance over the model of major gene alone or multifactorial inheritance alone. No significant heterogeneity could be detected between the high-incidence group (Oriental or Japanese) and the low-incidence group (non-Oriental) in the underlying general model, although higher heritability was observed in general. When families were classified into "severe" and "mild" phenotypes based on cleft lip vs. cleft lip and palate or unilateral vs. bilateral cleft in the proband, no significant differences could be detected between the two types in the underlying genetic model.     

Genetic basis of tobacco smoking: strong association of a specific major histocompatibility complex haplotype on chromosome 6 with smoking behavior

The genetic basis for addiction to tobacco smoking--particularly that of the perception of olfactory stimuli that may be important in reinforcing smoking addiction--is largely unknown. A cluster of genes for olfactory receptors is in close proximity to the MHC region on chromosome 6. Polymorphisms of MHC class III genes (RCCX modules, TNFA promoter polymorphisms) were determined in 101 healthy subjects and 232 coronary artery disease (CAD) patients from Hungary with defined tobacco smoking habits. A highly significant association between ever smoking (past + current smokers) and a specific MHC haplotype was observed (odds ratios = 2.14-4.13; P-values = 0.012 to <0.001). This haplotype is characterized by the presence of C4A null alleles and a solitary short C4B gene linked to the TNF2 allele of the promoter for TNFA gene. This haplotype occurred more frequently in the ever smokers than in the never smokers [odds ratio: 4.97 (1.96-12.62); P = 0.001], and such associations were stronger in women (odds ratio = 13.6) than in men (odds ratio = 2.79). An independent study of complement C4 protein polymorphism and smoking habits in Icelandic subjects (n = 351) yielded similar and confirmative results. Considering the documented link between olfactory stimuli and smoking in females, and the presence of a cluster of odorant receptor genes close to the MHC class I region, our findings implicate a potential role of the MHC-linked olfactory receptor genes in the initiation of smoking.     

Ascending spinal pathways for the somatosympathetic A and C reflexes.

Sympathetic unit nerve activity was recorded from the cervical sympathetic trunk of anesthetized, vagotomized, and carotid sinus-denervated cats. Single pulses or short trains of pulses with suprathreshold intensity for afferent C fibers were applied to the common peroneal nerve to elicit somatosympathetic A or simultaneous A and C reflexes, respectively. Bilateral lesions of an ascending spinal pathway in the dorsolateral sulcus area (DLS) of the T12 spinal cord eliminated the sympathetic C reflex. The remaining somatosympathetic A reflex was abolished by the subsequent lesion of an ascending spinal pathway in the dorsolateral funiculus (DLF) bilaterally. The duration of the silent period was found to be mainly influenced by an ascending spinal pathway in the DLF. It was concluded that excitation of afferent A fibers activates ascending pathways mainly in the DLF and a smaller contribution in the DLS of the spinal cord. This produces a small reflex excitation (A reflex) followed by a long silent period in the sympathetic nerves resulting in a net decrease of total sympathetic nerve activity. On the other hand, excitation of afferent C fibers activates the ascending spinal pathway in the DLS. This produces a reflex excitation (C reflex) which exceeds the decreased activity due to the silent period resulting in a net increase of total sympathetic nerve activity.     

Real-time PCR for quantitation of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in naturally-infected and challenged pigs

Real-time PCR assays were developed for quantitative detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2). The established real-time PCR for the quantitation of PRRSV cDNA and PCV2 DNA were found to be in the 9-log(10) linear dynamic range with excellent linearity and reliable reproducibility. Using these techniques, the distribution and quantitation of PRRSV and PCV2 in naturally infected and challenged pigs were investigated. The viral concentrations were expressed as the mean log(10) viral DNA or cDNA copy numbers per mg or ml of tested samples. For pigs infected naturally with both viruses, the lung, spleen, tonsil and lymphoid organs had the highest viral burdens with ranges from 5.73 to 8.38 and 5.65 to 6.91 for PRRSV and PCV2, respectively. The injection of formalin-inactivated Salmonella choleraesuis emulsified in complete Freund's adjuvant 1 week before and after the inoculation of both viruses resulted in PRRSV replication enhancement 2 weeks post-challenge. However, this facilitated the clearance of PRRSV 4 weeks post-challenge. Results from this study show that the established quantitative PCR could be a useful tool when applied to vaccine development and pathogenesis studies in the future.     

Role of predicted transmembrane domains for type III translocation, pore formation, and signaling by the Yersinia pseudotuberculosis YopB protein

YopB is a 401-amino-acid protein that is secreted by a plasmid-encoded type III secretion system in pathogenic Yersinia species. YopB is required for Yersinia spp. to translocate across the host plasma membrane a set of secreted effector proteins that function to counteract immune signaling responses and to induce apoptosis. YopB contains two predicted transmembrane helices (residues 166 to 188 and 228 to 250) that are thought to insert into the host plasma membrane during translocation. YopB is also required for pore formation and host-cell-signaling responses to the type III machinery, and these functions of YopB may also require membrane insertion. To elucidate the importance of membrane insertion for YopB function, YopB proteins containing helix-disrupting double consecutive proline substitutions in the center of each transmembrane domain were constructed. Yersinia pseudotuberculosis strains expressing the mutant YopB proteins were used to infect macrophages or epithelial cells. Effector translocation, pore formation, and host-cell-signaling responses were studied. Introduction of helix-disrupting substitutions into the second transmembrane domain of YopB resulted in a nonfunctional protein that was not secreted by the type III machinery. Introduction of helix-disrupting substitutions into the first transmembrane domain of YopB resulted in a protein that was fully functional for secretion and for interaction with YopD, another component of the translocation machinery. However, the YopB protein with helix-disrupting substitutions in the first transmembrane domain was partially defective for translocation, pore formation, and signaling, suggesting that all three functions of YopB involve insertion into host membrane.     

Leukocyte apoptosis and its significance in sepsis and shock

Sepsis and multiple organ failure continue to be significant problems among trauma, burn, and the critically ill patient population. Thus, a number of laboratories have focused on understanding the role of altered apoptotic cell death in contributing to immune and organ dysfunction seen in sepsis and shock. Immune cells that undergo altered apoptotic changes include neutrophils, macrophages, dendritic cells, as well as various lymphocyte populations. Evidence of epithelial as well as endothelial cell apoptotic changes has also been reported. Although mediators such as steroids, tumor necrosis factor, nitric oxide, C5a, and Fas ligand (FasL) appear to contribute to the apoptotic changes, their effects are tissue- and cell population-selective. As inhibiting Fas-FasL signaling (e.g., gene deficiency, Fas fusion protein, or Fas short interfering RNA administration), caspase inhibition (caspase mimetic peptides), and/or the overexpression of downstream antiapoptotic molecules (e.g., Bcl-2, Akt) improve survival of septic mice, it not only demonstrates the pathological significance of this process but points to novel targets for the treatment of sepsis.     

Use of Monoclonal Antibodies That Recognize p60 for Identification of Listeria monocytogenes

Listeria monocytogenes causes major food-borne outbreaks of disease worldwide. Specific identification of this microorganism is of utmost importance to public health and industry. Listeria species are known to secrete a 60-kDa protein collectively termed p60, which is encoded by the iap (invasion-associated protein) gene and secreted in large quantities into the growth media. p60 is a highly immunogenic murein hydrolase that is essential for cell division. Due to these properties, p60 is an ideal diagnostic target for the development of immunological detection systems for L. monocytogenes. We report here two independent lines of monoclonal antibody (MAb): p6007, which specifically recognizes L. monocytogenes p60, and p6017, which reacts with a wide range of Listeria p60 proteins. By combining these antibodies with a polyclonal antibody, we developed efficient sandwich enzyme-linked immunosorbent assay (ELISA) systems which can specifically identify L. monocytogenes or generally detect Listeria species. Since an excess amount of the peptide corresponding to PepA or PepD did not interfere with the ELISA, and direct ELISAs were unable to detect both peptides, we concluded that the epitope presumed to be recognized by p6007 or p6017 could be distinguished from PepA and PepD as described by Bubert et al. (Appl. Environ. Microbiol. 60:3120-3127, 1997). To our best knowledge, this is the first example of an immunological identification system that uses p60-recognizing MAbs.     

The effect of alphaMSH analogues on rat bones

Melanocortin is the downstream mediator of leptin signaling and absence of leptin signaling in ob/ob and db/db mice revealed the enhancement of bone formation through the central regulation. While alpha-melanocyte-stimulating hormone (alphaMSH) inhibits the secretion of interleukin-1alpha and tumor necrosis factor-alpha from the inflammatory cells, alphaMSH can also enhance clonal expansion of pro B cells linked to stimulation of osteoclastogenesis. Therefore, we tested the effect of melanocortin on bones. alphaMSH analogues [(6)His]alphaMSH-ND and [(6)Asn]alphaMSH-ND were synthesized and the radio-ligand receptor binding- and cyclic AMP generating activity were analyzed in China Hamster Ovary cell line over- expressing melanocortin receptors. The EC(50) of [(6)His]alphaMSH-ND measured from melanocortin-1, 3, 4 and 5 receptors were 0.008 +/- 0.0045, 1.523 +/- 0.707, 0.780 +/- 0.405, and 250.320 +/- 42.234 nM, respectively, and the EC(50) of [(6)Asn]alphaMSH-ND were 16.8 +/- 6.94, 271.8 +/- 21.95, 8.0 +/- 1.21, and 1132.5 +/- 635.46 nM, respectively. Four weeks after the subcutaneous injection of the analogues, the body weights in the [(6)His]alphaMSH-ND and the [(6)Asn]alphaMSH-ND treated groups (346.0 +/- 20.63 g vs. 350.0 +/- 13.57 g) were lower than that of the vehicle treated group (375.8 +/- 17.31 g, p < 0.05). There was no difference in the total femoral BMD measured by dual x-ray absorptiometry among the three groups. Among the three groups, there were no differences in the total numbers of crystal violet positive- or alkaline phosphatase positive colonies, in the expression of Receptor Activator of Nuclear Factor Kappa-B ligand on the tibia and the total number of multinucleated osteoclast-like cells differentiated from primary cultured bone marrow cells. From the above results, no evidence of bone gain or loss was found after treatment of the alphaMSH analogues peripherally.     

The value of immunohistochemical detection of P-glycoprotein in breast cancer before and after induction chemotherapy.

We have studied the patterns of P-glycoprotein expression before and after 3 cycles of induction chemotherapy (5-fluorouracil, adriamycin and cyclophosphamide) using immunohistochemically stained paraffin-embedded specimen of 28 patients with locally advanced breast cancer. The frequency of P-glycoprotein expression in untreated breast cancer turned out to be very low: only one out of 28 untreated, biopsy specimen at the time of diagnosis was positive. The frequency of P-glycoprotein expression was markedly increased from 9.1% before chemotherapy to 63.6% after induction chemotherapy (p = 0.006). After 3 cycles of induction chemotherapy, 25 patients had obtained clinical response to chemotherapy (4, CR; 21, PR). Eleven out of 25 tumors (44%) showing clinical response and all three tumors (100%) with minimal response have expressed P-glycoprotein. One out of 6 patients (16.7%) with microscopic residual tumor seen in mastectomy specimen expressed P-glycoprotein, whereas 13 of 22 patients (59.1%) with gross residual tumor showed the presence of P-glycoprotein (p = 0.08). The frequency of intrinsic P-glycoprotein expression in untreated breast cancer was quite low, but approximately half of the patients do acquire P-glycoprotein expression during the cycles of induction chemotherapy. Therefore, the results suggest that the immunohistochemical detection of P-glycoprotein on residual tumor cells after induction chemotherapy can predict acquired drug resistance in breast cancer.