Dual innervation of the mylohyoid muscle by the trigeminal and hypoglossal nerves: A case report.

During the routine dissection of a cadaveric specimen, the left mylohyoid muscle was found to be innervated by both the trigeminal and hypoglossal nerves. This variation was found unilaterally. To our knowledge this dual innervation of the mylohyoid muscle is an extremely rare variation. The possibility of these variants may lead to clinical consequences such as anesthesia failure and iatrogenic injury during surgical procedures in this region. We discuss this anatomical variation and possible developmental etiologies.

Toward High Precision XCO2 Retrievals From TanSat Observations: Retrieval Improvement and Validation Against TCCON Measurements.

TanSat is the 1st Chinese carbon dioxide (CO2) measurement satellite, launched in 2016. In this study, the University of Leicester Full Physics (UoL-FP) algorithm is implemented for TanSat nadir mode XCO2 retrievals. We develop a spectrum correction method to reduce the retrieval errors by the online fitting of an 8th order Fourier series. The spectrum-correction model and its a priori parameters are developed by analyzing the solar calibration measurement. This correction provides a significant improvement to the O2 A band retrieval. Accordingly, we extend the previous TanSat single CO2 weak band retrieval to a combined O2 A and CO2 weak band retrieval. A Genetic Algorithm (GA) has been applied to determine the threshold values of post-screening filters. In total, 18.3% of the retrieved data is identified as high quality compared to the original measurements. The same quality control parameters have been used in a footprint independent multiple linear regression bias correction due to the strong correlation with the XCO2 retrieval error. Twenty sites of the Total Column Carbon Observing Network (TCCON) have been selected to validate our new approach for the TanSat XCO2 retrieval. We show that our new approach produces a significant improvement on the XCO2 retrieval accuracy and precision when compared to TCCON with an average bias and RMSE of -0.08 ppm and 1.47 ppm, respectively. The methods used in this study can help to improve the XCO2 retrieval from TanSat and subsequently the Level-2 data production, and hence will be applied in the TanSat operational XCO2 processing.

Adaptive response in embryogenesis: vi. Comparative microarray analysis of gene expressions in mouse fetuses.

PURPOSE: Exposure of sublethal doses of ionizing radiation can induce protective mechanisms against a subsequent higher dose irradiation. This phenomenon, called radiation-induced adaptive response (AR), has been described in a wide range of biological models. We previously demonstrated the existence of AR in mice during late organogenesis. In this study, we investigated molecular mechanisms underlying AR in this model. MATERIALS AND METHODS: Using DNA microarrays, we performed a global analysis of transcriptome regulations in adapted and non-adapted cells collected from whole mouse fetuses, after in utero exposure to priming irradiation. RESULTS: We identified AR-specific gene modulations. Our results suggested the involvement of signal transduction and Tumor protein (p53)-related pathways in the induction of AR. CONCLUSIONS: Our results are in agreement with previous investigations showing that AR could be dependant on p53 activity. The observed gene modulations may also have possible consequences for subsequent developmental process of the fetus. This is the first report of AR-specific modulations at the molecular level in utero, which could serve as a basis for subsequent studies aimed at understanding AR in this model and possible long-term effects.

Human leukocyte histocompatibility antigen class II-induced cytokines from human gingival fibroblasts promote proliferation of human umbilical vein endothelial cells: potential association with enhanced angiogenesis in chronic periodontal inflammation.

BACKGROUND AND OBJECTIVE: The role of human leukocyte histocompatibility antigen (HLA) class II molecules on non-antigen-presenting cells has been a matter of controversy. We previously reported that HLA-II molecules on human gingival fibroblasts (GF) do not present antigens, but transduce signals into the cells, resulting in the expression of several cytokines, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), regulated upon activation, normal T-cell expressed and secreted (RANTES) and IL-8. However, the exact role of these cytokines, as well as other cytokines which are potentially secreted from GF, in the pathogenesis of chronic periodontal inflammation is not fully understood. The aim of this study was to observe the effects of HLA-II-induced cytokines on the proliferation of human umbilical vein endothelial cells (HUVEC). MATERIAL AND METHODS: Antibody-based cytokine-microarray analyses were performed to detect potential cytokines associated with angiogenesis. Next, cytokine productivity was confirmed by quantitative methods. Then, cell proliferation assay was performed to see whether these cytokines promoted the proliferation of HUVEC. RESULTS: Besides IL-6, MCP-1, RANTES and IL-8, growth-related gene product (GRO) was newly identified as an HLA-II-induced cytokine released from GF. This was confirmed by a quantitative method. Cell culture supernatant from HLA-II-stimulated GF cultures promoted the growth of HUVEC. Addition of anti-IL-8 neutralizing antibody, anti-CXC receptor (CXCR)1 antibody and anti-MCP-1 antibody inhibited the growth of HUVEC in a dose-dependent manner, while addition of anti-GROalpha antibody did not. CONCLUSION: The HLA-II-induced IL-8, via CXCR1, as well as MCP-1 from GF, promotes endothelial cell proliferation, which is possibly associated with enhanced angiogenesis in chronic periodontal lesions.

p12(DOC-1) is a novel cyclin-dependent kinase 2-associated protein.

Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12(DOC-1) is a growth suppressor isolated from normal keratinocytes. We report that p12(DOC-1) associates with CDK2. More specifically, p12(DOC-1) associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12(DOC-1) resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12(DOC-1) transfectants ( upward arrow G(1) and downward arrow S). The p12(DOC-1)-mediated decrease of CDK2 was prevented if the p12(DOC-1) transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin beta-lactone, suggesting that p12(DOC-1) may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12(DOC-1)-mediated, CDK2-associated cell cycle phenotypes. These data support p12(DOC-1) as a specific CDK2-associated protein that negatively regulates CDK2 activities by sequestering the monomeric pool of CDK2 and/or targets CDK2 for proteolysis, reducing the active pool of CDK2.

Oral cancer in vivo gene expression profiling assisted by laser capture microdissection and microarray analysis.

Large scale gene expression profiling was carried out on laser capture microdissected (LCM) tumor and normal oral epithelial cells and analysed on high-density oligonucleotide microarrays. About 600 genes were found to be oral cancer associated. These oral cancer associated genes include oncogenes, tumor suppressors, transcription factors, xenobiotic enzymes, metastatic proteins, differentiation markers, and genes that have not been implicated in oral cancer. The database created provides a verifiable global profile of gene expression during oral carcinogenesis, revealing the potential role of known genes as well as genes that have not been previously implicated in oral cancer.

Polymorphism of the 5' flanking region of the IL-12 receptor beta2 gene partially determines the clinical types of leprosy through impaired transcriptional activity.

BACKGROUND: Individual differences in T cell responsiveness to interleukin 12 (IL-12), resulting from inherited factors, may be responsible for differences in the intensity of cell mediated immune (CMI) responses in patients with leprosy, a disease with a wide clinical spectrum. AIM: Polymorphisms in the 5' flanking region of the IL12RB2 gene were analysed to determine potential immunogenetic factors affecting CMI responses, using leprosy as a model. METHODS: Polymorphisms in the 5' flanking region of IL12RB2 were examined using direct sequencing techniques, and allele frequencies between patients with lepromatous leprosy and patients with tuberculoid leprosy were compared. The effect of these single nucleotide polymorphisms (SNPs) on IL12RB2 expression was estimated using the dual luciferase reporter gene assay in Jurkat T cells. RESULTS: Several SNPs, including -1035A>G, -1023A>G, -650delG, and -465A>G, were detected within the 5' flanking region of IL12RB2. The frequency of haplotype 1 (-1035A, -1023A, -650G, -464A) was high in the general Japanese population, but was significantly lower in lepromatous patients compared with tuberculoid patients and healthy controls. Reporter gene assays using Jurkat T cells revealed that all haplotypes carrying one or more SNP exhibited a lower transcriptional activity compared with haplotype 1. CONCLUSION: SNPs within the 5' flanking region of IL12RB2 affect the degree of expression of this gene and may be implicated in individual differences in CMI responsiveness to mycobacterial antigens, leading to lepromatous or tuberculoid leprosy.

Reduction of p12DOC-1 expression is a negative prognostic indicator in patients with surgically resected oral squamous cell carcinoma.

PURPOSE: p12DOC-1 is a growth suppressor that negatively regulates cyclin-dependent kinase 2 (CDK2) activities. Expression of p12DOC-1 is reduced and/or lost in tumor tissues. The purpose of this study is to correlate in vivo the expression of p12DOC-1 in oral cancer tissues by immunohistochemistry with clinical and pathological parameters. EXPERIMENTAL DESIGN: Twenty-five cases of normal oral mucosa and 127 cases of oral squamous cell carcinomas were evaluated. Patients' charts were reviewed for clinical, pathological, and 10-year survival data. Because p12DOC-1 is a growth suppressor and associates with CDK2, parallel immunostaining was done for proliferating cell nuclear antigen and CDK2 to evaluate cell proliferation and potential correlation with CDK2. RESULTS: Our results showed that strong p12DOC-1 staining was uniformly seen in normal oral mucosa. p12DOC-1 staining was reduced or absent in 81 cases (63.8%) of oral squamous cell carcinomas. Decreased p12DOC-1 staining (<25% of cells stained) correlated with tumor mode of invasion (P = 0.001) and higher proliferating cell nuclear antigen (P = 0.0028) and CDK2 (P = 0.0020) expression. Survival analysis showed significant correlation of low p12DOC-1 expression with the risk of cervical lymph node metastasis (P = 0.001) and patients' 10-year survival status (P = 0.0214). CONCLUSIONS: These results allow us to conclude that reduction of p12DOC-1 protein expression is a frequent event in oral cancers. Intratumor immunohistochemical evaluation of p12DOC-1 expression can be an adjunctive prognostic indicator for patients with oral cancer.

Angiotensin-converting enzyme inhibition with enalapril slows progressive intima-media thickening of the common carotid artery in patients with non-insulin-dependent diabetes mellitus.

BACKGROUND AND PURPOSE: Whether angiotensin-converting enzyme (ACE) inhibitors have any clinically significant antiatherogenic effects in humans remains unproven. We undertook a prospective randomized clinical trial of 98 patients with non-insulin-dependent diabetes mellitus (NIDDM) to examine the efficacy of ACE inhibition with enalapril for preventing intima-media (IM) thickening of the carotid wall as measured ultrasonographically. METHODS: Ninety-eight NIDDM patients were randomly assigned either to enalapril at 10 mg/d (n=48) or to a control group (n=50); the planned duration of the trial was 2 years. All patients were seen at baseline (study entry) and 2 subsequent formal annual evaluations, in addition to standard clinical management for NIDDM. IM thickening and vascular lumen diameters were determined for all patients on the basis of baseline and 2 subsequent annual evaluations with carotid ultrasonography. We performed an intent-to-treat analysis to assess changes in IM thickening over the course of the study. RESULTS: Annual IM thickening measurements of the right and left common carotid arteries were 0.01+/-0.02 and 0.01+/-0.02 mm/y in the enalapril-treated group and 0.02+/-0.03 and 0.02+/-0.02 mm/y in the control group, respectively (P<0.05). From regression analysis, annual IM thickening was found to be predicted by enalapril use, sex, and insulin use (F(3,94)=3.86, P=0.012). When we controlled for these other variables, enalapril use reduced annual IM thickening of right and left common carotid arteries by 0.01+/-0.004 mm/y relative to the control group over the course of this study. CONCLUSIONS: Long-term treatment with an ACE inhibitor (enalapril) slows progressive IM thickening of the common carotid artery in NIDDM patients.

Duration threshold of induced hypertension on cerebral blood flow, energy metabolism, and edema after transient forebrain ischemia in gerbils.

We have investigated whether there is a duration threshold for the effects of phenylephrine-induced hypertension on CBF, brain energy metabolism, and cerebral parenchymal specific gravity (SG) following transient forebrain ischemia in gerbils. Sixty gerbils were randomly assigned to one of the four treatment groups: one control group and three groups subjected to an increase of 25 mm Hg in MABP induced by treatment, 30 min after reperfusion, with phenylephrine for 15 min, 30 min, or 60 min. The local CBF was measured continuously, and the SG was evaluated 120 min after reperfusion. Sequential changes in brain energy metabolism, as shown by the ratio of phosphocreatine to inorganic phosphate (Pi), the beta-ATP/Pi ratio, and intracellular pH, were also measured. The 15-min induced hypertension regimen was most suited to the recovery of brain energy metabolism, which was associated with an increase in local CBF and a decrease in cerebral edema. These results demonstrate that a suitable duration can be chosen to optimize the beneficial effects of phenylephrine-induced hypertension on ischemic brain injury following transient forebrain ischemia.

Phosphatidylserine induces apoptosis in CHO cells without mitochondrial dysfunction in a manner dependent on caspases other than caspases-1, -3, -8 and -9.

Treatment of Chinese hamster ovary K1 cells with phosphatidylserine (PS) caused typical apoptosis with distinct morphological and biochemical features in a dose- and time-dependent manner. However, unlike camptothecin-induced apoptosis, changes in mitochondrial transmembrane potential were not observed. In addition, cytochrome c release did not occur in PS-induced apoptosis. A pan caspase inhibitor, Z-VAD, significantly inhibited the apoptosis, but inhibitors of caspase-1, -3, -8 and -9 did not. Activities of caspase-1, -3, -8 and -9 were increased by treatment of the cells with camptothecin, but not with PS. These results suggest that PS-induced apoptosis occurs without the collapse of mitochondrial transmembrane potential and without the release of cytochrome c, in a manner independent of caspase-1, -3, -8 and -9.

Optimized conditions for gene transfection into the human eosinophilic cell line EoL-1 by electroporation.

Eosinophils are emerging as an increasingly important cell in the immunoregulatory network of normal and pathological processes. No studies has yet described optimized experimental strategies to transfect DNA into human eosinophils. Using a frequently employed in vitro model of human eosinophil, the EoL-1 cells, we now described the optimal transfection of DNA into these cells by electroporation. Our results indicate that electroporation can efficiently and reproducibly transfect DNA into EoL-1 cells. Optimal electroporation conditions consist of the use of 1 X RPMI medium 1640 with 10% FBS, voltage setting at 275 V, 1150 microF capacitance, 40 mg of DNA and 4.0 X 10(7) cells/ml per electroporation in a total volume of 0.5 ml in 0.4 cm gap cuvettes. These conditions may be a useful protocol for transfecting eosinophil cell lines.

Laser capture microdissection-generated target sample for high-density oligonucleotide array hybridization.

Current advances in biomolecular technology allow precise genetic fingerprinting of specific cells responsible for the pathogenesis of human diseases. This study demonstrates the feasibility of generating target samples from laser capture microdissection (LCM) tissues suitable for hybridization of high-density oligonucleotide arrays for gene expression profiling. RNA was successfully isolated by LCM from three paired specimens of oral cancer and linearly amplified using T7 RNA polymerase. Evaluation of the cDNA revealed that five of five cellular maintenance transcripts are detected. Biotinylated cRNA was generated and hybridized to the human Test 1 GeneChip probe arrays, which demonstrated that the RNA is of sufficient quality and integrity to warrant further analysis. Subsequent hybridization of the samples to the HuGenFL GeneChip probe arrays revealed that 26.5%-33.0% of the approximately 7000 represented genes are expressed in each of the six samples. These results demonstrate that LCM-generated tissues can generate sufficient quality cRNA for high-density oligonucleotide microarray analysis, an important step in determining comprehensive gene expression profiling using this high-throughput technology.

T cell responses to 53-kDa outer membrane protein of Porphyromonas gingivalis in humans with early-onset periodontitis.

Patients with early-onset periodontitis (EOP) are susceptible to infection with periodontopathic bacteria, such as Porphyromonas gingivalis. Ag53, 53-kDa outer membrane protein of P. gingivalis, evokes strong humoral immune responses in EOP patients. In a first step to clarify how host immune cells recognize Ag53, we established Ag53-specific short-term T cell lines from 22 subjects including 6 EOP patients and 16 healthy donors, using overlapping peptides based on Ag53 amino acid sequences. All T cell lines from active EOP patients recognized a common region (p141-181, especially p141-161) on Ag53, while those from healthy donors showed heterogeneous specificity. p141-181 was not recognized by T cell lines established from EOP patients following therapy. A monoclonal antibody to HLA-DRB 1 inhibited Ag53-induced proliferation of most of the T cell lines. Our observations suggest that, although antigen-presenting molecules are common in EOP patients and in healthy individuals, p141-161 includes a major T cell epitope(s) on Ag53 for active EOP patients but not for healthy individuals or inactive EOP patients.

Efficacy of traditional herbal medicines in combination with acyclovir against herpes simplex virus type 1 infection in vitro and in vivo.

Traditional herbal medicines have been safely used for the treatment of various human diseases since ancient China. We selected 10 herbal extracts with therapeutic antiherpes simplex virus type 1 (HSV-1) activity. Among these, Geum japonicum Thunb., Rhus javanica L., Syzygium aromaticum (L.) Merr. et Perry, or Terminalia chebula Retzus showed a stronger anti-HSV-1 activity in combination with acyclovir than the other herbal extracts in vitro. When acyclovir and/or a herbal extract were orally administered at doses corresponding to human use, each of the 4 combinations significantly limited the development of skin lesions and/or prolonged the mean survival times of infected mice compared with both acyclovir and the herbal extract alone (P < 0.01 or 0.05). These combinations were not toxic to mice. They reduced virus yields in the brain and skin more strongly than acyclovir alone and exhibited stronger anti-HSV-1 activity in the brain than in the skin, in contrast to acyclovir treatment by itself. Combinations of acyclovir with historically used herbal medicines showed strong combined therapeutic anti-HSV-1 activity in mice, especially reduction of virus yield in the brain.

Prophylactic efficacy of traditional herbal medicines against recurrent herpes simplex virus type 1 infection from latently infected ganglia in mice.

Traditional herbal medicines with anti-herpes simplex virus type 1 (HSV-1) activity in vivo were examined for their prophylactic effects on recurrent HSV-1 infection in mice. Mice were intradermally infected with HSV-1 in the pinna and recurrent HSV-1 disease was induced by ultraviolet irradiation. Herbal extracts arrested the progression of recurrent HSV-1 disease, reduced the incidence of severe erythema and/or vesicles in the pinna, and/or shortened the period of severe recurrent lesions compared with water-administered mice (P < 0.01 or 0.05). Similarly, the prophylactic treatment of herbal extracts limited the development of recurrent skin lesions induced by stripping with cellophane tape physically. The prophylactic efficacy on recurrence was confirmed by the absence of HSV DNA in the skin lesions. HSV-1 genome was revealed to exist in the trigeminal ganglia but not in the pinna of latently infected mice before stimuli by a nested-polymerase chain reaction assay. After stimuli, HSV-1 genome was detected in both pinna and trigeminal ganglia of latently infected mice administered with water. However, prophylactic treatment decreased the rate of detection of HSV-1 genome in the stimulated pinna. Thus, the herbal extracts exhibited prophylactic efficacy against recurrent HSV-1 disease in mice and modulated the recurrent HSV-1 infection.

Collagenase of human skin basal cell epithelioma.

Collagenase of human basal cell epithelioma was purified by sequential ammonium sulfate precipitation, Sephadex gel filtration and affinity chromatography on collagen-polyacrylamide gel. The collagenase, when partially purified, was found to have an approximate molecular weight of 50,000. The purified enzyme contained no caseinolytic activity. On polyacrylamide gel electrophoresis, the purified enzyme gave a single protein band. The purified collagenase cleaved native acid-soluble guinea pig skin collagen at 37 degrees C with a pH optimum of 8. The enzyme was inhibited by EDTA, cysteine, and human serum but not by soybean trypsin inhibitor. Heparin did not stimulate the enzyme activity. Purified collagenase reduced the specific viscosity of native acid-soluble guinea pig skin collagen to 50 per cent of its original value at 27 degrees C. Polyacrylamide gel disc electrophoresis of the reaction products showed bands corresponding to alphaA, betaA, and alphaB fragments. Electron microscopic examination of SLS aggregates of the reaction products showed that the cleavage site by the enzyme was at a point 75 per cent from the "A" end (TCA75) and 25 per cent from the "B" end (TCB25) of the collagen molecule.

Prevention of interphase death in rat thymocytes by bisulfite.

The effect of sodium bisulfite, a specific inhibitor of chromatin proteolysis, on radiation damage in rat thymocytes in vitro was examined. Rat thymocytes irradiated with 1 kR X rays in vitro were incubated at 37 degrees C with 10 mM glucose for 4 to 6 hr. During that time development of interphase death as judged by erythrosin B uptake, release of low molecular weight DNA (free DNA), and reduction in cell size was measured. Sodium bisulfite added to the cells at the beginning of incubation exerted a marked preventive effect on radiation damage. The effect was enhanced with increasing concentration of bisulfite from 0.25 to 2 mM. The effect of bisulfite was reversible; i.e., removal of bisulfite from the cells resulted in the reappearance of the radiation damage.

Collagenolytic activities of cultured human malignant melanoma cells.

Three human malignant melanomas were cultured in pure populations and one tumor was cloned into melanotic and amelanotic cell lines. In the homogenates of these cultured cells, specific collagenase activities were demonstrated by isotope release from 14C-labeled collagen, disc electrophoresis, and specific cleavage of collagen molecules as demonstrated in the segment long spacing form. No significant collagenase activity was observed in the culture media. Interestingly, early cultures had a high collagenase activity in the cells and as they were successively subcultured, the activity diminished. Cysteine completely inhibited the degradation of tropocollagen as determined by disc electrophoresis and EDTA partially inhibited the degradation. It is concluded that human malignant melanoma cells produce a specific collagenase in vitro which can be extracted in early culture directly from the homogenate.

Radiation-induced formation of apoptotic bodies in rat thymus.

The process of interphase death of thymocytes in whole-body X-irradiated rats were studied. Cell size distribution analysis indicates that cell fragments (= apoptotic bodies) appeared in the thymus and increased in number depending on dose (200-1000 R) and time (2-6 hr) after irradiation with corresponding decrease in normal-size thymocytes. Occurrence of nuclear fragmentation in association with the cellular fragmentation was proved with cytofluorometric determination of DNA content in individual cells. Scanning electron microscopic observations also revealed extensive fragmentation of cells in the irradiated rat thymus. The results show clearly that cells as well as nuclei fragment rapidly into smaller pieces of various sizes in the irradiated rat thymus as commonly observed with apoptosis.