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PubMed1990

The circadian rhythm of intra-acinar profiles of alcohol dehydrogenase activity in rat liver: a microquantitative study.

Using microquantitative measurements of alcohol dehydrogenase activity in microdissected samples of liver tissue along the sinusoidal length, the intra-acinar distribution profiles were studied in seven groups of female rats at different times during 24 h with a light phase from 6:30 h to 18:30 h. The mean values of alcohol dehydrogenase activity showed a circadian rhythm with a minimum at 13.30 h and a maximum at 17.30 h (p less than 0.001). However, the intra-acinar gradients remained almost unchanged, indicating that increase and decrease in enzyme activity takes place simultaneously in all parts of the liver acinus. This observation, together with data from the literature, suggests that the circadian rhythm of alcohol dehydrogenase activity reflects variations in different liver cell constituents, rather than enzyme protein synthesis or proteolysis.

Alcohol Dehydrogenase↗
PubMedDate not supplied

Methods for the study of liver cell heterogeneity.

A large number of histological, histochemical and biochemical techniques are available for studying liver cell heterogeneity. Structural differences are recognized by morphometric analyses of electron micrographs. The zonal heterogeneity of enzyme activities can be demonstrated by histochemistry and more precisely by ultramicrobiochemical assays in microdissected periportal and perivenous tissue. Immunohistochemistry is useful for quantifying and localizing proteins, especially isoenzymes, without depending on their biological activity. The zonal quantification of specific mRNA can be achieved by in situ hybridization. The different structural and enzymic equipment of periportal and perivenous tissue found by these techniques has led to the concept of metabolic zonation. This hypothesis can be confirmed by determination of metabolic rates in perfused liver after selective zonal damage, in separated periportal and perivenous hepatocytes as well as in periportal and perivenous tissue of perfused liver by non-invasive techniques.

Animals↗
PubMed1987

The study of the effect of human chorionic gonadotrophic (HCG) hormone on the survival of adrenal medulla transplant in brain. Preliminary study.

Autologous adrenal cortex was completely dissected off the medulla using microdissection. The adrenal medulla tissue was transplanted to the lateral ventricle in twenty-one adult rats. Group A consisting of ten animals received 200 units of HCG per 100 gram body weight daily for one week and then on every alternate day for seven weeks. The remaining rats (group B) served as control. After eight weeks the brains were removed and a volumetric study was done using histological sections. High pressure liquid chromatography (HPLC) with electrochemical detection for catecholamine assay of the graft was also undertaken. The graft survival in group A was significantly higher (p less than 0.005) than group B. The level of norepinephrine was significantly higher in group A while dopamine was higher in group B. The higher volume of graft tissue in group A suggests that HCG may help in increased survival and growth of the transplanted tissue. Higher levels of norepinephrine in group A would suggest a tendency for such grafts to be active to a greater extent with the treatment of HCG.

Adrenal Medulla↗
PubMed2004

VIP receptor antagonists inhibit mammary carcinogenesis in C3(1)SV40T antigen mice.

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist on mammary carcinogenesis were investigated using the C3(1)SV40T antigen (ag) mice. Ten microg/day VIPhybrid (VIPhyb) administered daily subcutaneously increased significantly the survival of C3(1)SV40Tag mice. At 5.2 months, VIPhyb significantly reduced the mammary tumor burden in C3(1)SV40Tag mice relative to control animals. 125I-VIP bound with high affinity to mouse mammary tumor homogenate. Because (Lys15, Arg16, Leu27)VIP1-7GRF8-27 (VPAC1 selective) but not Ro25-1553 (VPAC2 selective) inhibited specific 125I-VIP binding to mammary tumor membranes with high affinity, VPAC1 receptors predominate. By RT-PCR, VPAC1 receptor mRNA was detected in mammary tumors. By Western blot, a major 60 Kdalton band was detected in mammary tumor extracts using VPAC1 receptor antisera. By immunocytochemistry, VPAC1-R immunostaining was detected in the cytosol and plasma membrane but not the nucleus of fixed mammary tumor tissue. Using laser capture microdissected tumor cells and surface enhanced laser desorption/ionization (SELDI) techniques on mammary tumor cells, the proteomic profile was altered in mice treated with VIPhyb. Because VPAC1 receptor antagonists increase the survival and reduce the tumor burden in C3(1)SV40Tag mice, they may function as chemopreventive agents in mammary cancer.

Animals↗
PubMed2005

Expression of neural connexins and pannexin1 in the hippocampus and inferior olive: a quantitative approach.

Electrical synapses (or neuronal gap junctions) are thought to be essential for the generation of synchronous oscillatory activities in various areas of the brain. In this study, we quantified the steady state mRNA expression levels of two neuronal gap junction proteins, connexin36 (Cx36) and connexin45 (Cx45), as well as of pannexin1, a member of a novel class of communicative junction forming proteins, and of connexin47 (Cx47) which is expressed in oligodendrocytes. The expression levels of these genes were compared in two regions known for oscillatory activity and which are equipped with electrically coupled neurons. Assessment of the levels of mRNA expression in the hippocampus and the nuclear complex of the inferior olive (IO) was achieved by means of laser microdissection (LMM) in combination with real time RT-PCR. Our results demonstrate the differential expression of Cx36, Cx45, pannexin1 and Cx47 in the hippocampus, with pannexin1 showing the highest level of expression followed by Cx36, Cx47, and Cx45. In the IO, pannexin1 showed a comparable expression level as in the hippocampus, but connexin expression levels were increased. Upon direct comparison, the combination of LMM and real time RT-PCR data generated specific, robust and reproducible results consistent with recent data reported about connexin expression in the nervous system. We conclude that the analytical strategy shown here provides a technological solution to overcome the less sensitive and notoriously less specific analysis of connexin expression by in situ hybridization.

Animals↗
PubMed2005

Assessment of differential gene expression in vestibular epithelial cell types using microarray analysis.

Current global gene expression techniques allow the evaluation and comparison of the expression of thousands of genes in a single experiment, providing a tremendous amount of information. However, the data generated by these techniques are context-dependent, and minor differences in the individual biological samples, methodologies for RNA acquisition, amplification, hybridization protocol and gene chip preparation, as well as hardware and analysis software, lead to poor correlation between the results. One of the significant difficulties presently faced is the standardization of the protocols for the meaningful comparison of results. In the inner ear, the acquisition of RNA from individual cell populations remains a challenge due to the high density of the different cell types and the paucity of tissue. Consequently, laser capture microdissection was used to selectively collect individual cells and regions of cells from cristae ampullares followed by extraction of total RNA and amplification to amounts sufficient for high throughput analysis. To demonstrate hair cell-specific gene expression, myosin VIIA, calmodulin and alpha9 nicotinic acetylcholine receptor subunit mRNAs were amplified using reverse transcription-polymerase chain reaction (RT-PCR). To demonstrate supporting cell-specific gene expression, cyclin-dependent kinase inhibitor p27kip1 mRNA was amplified using RT-PCR. Subsequent experiments with alpha9 RT-PCR demonstrated phenotypic differences between type I and type II hair cells, with expression only in type II hair cells. Using the laser capture microdissection technique, microarray expression profiling demonstrated 408 genes with more than a five-fold difference in expression between the hair cells and supporting cells, of these 175 were well annotated. There were 97 annotated genes with greater than a five-fold expression difference in the hair cells relative to the supporting cells, and 78 annotated genes with greater than a five-fold expression difference in the supporting cells relative to the hair cells.

Acoustic Maculae↗
PubMed2004

Altered expression and loss of heterozygosity of the LOT1 gene in ovarian cancer.

OBJECTIVES: Previously, we demonstrated that the LOT1 (PLAGL1/ZAC1) gene encodes a zinc-finger transcription factor and has growth suppressive effects in carcinoma cell lines. The gene is localized on chromosome 6q24-25, a common site for loss of heterozygosity (LOH) in many solid tumors including ovarian cancer. In this study, we evaluated the LOT1 gene expression and allelic deletion in the tumor tissues in order to provide additional evidence to support the gene's potential role in cancer. METHODS: The LOT1 gene expression was analyzed in malignant ovarian epithelium obtained from frozen human ovarian tumor tissues using laser capture microdissection (LCM) and real-time PCR techniques. Highly frequent single nucleotide polymorphic (SNP) sites within the LOT1 gene were identified and used for PCR and direct sequencing to determine the occurrence of allelic imbalance in a series of surgically resected ovarian and breast carcinomas. RESULTS: The analysis revealed that LOT1 mRNA expression was not detectable in 12 of 31 (39%) cases of ovarian cancer and was variable between the remaining 19 tumor specimens. These findings are consistent with the previous data that showed altered expression of LOT1 in different human ovarian carcinoma cell lines. In addition, we analyzed the occurrence of LOT1 allelic deletion in different ovarian tumor genomic DNA samples that included papillary serous (majority), mucinous, and primary peritoneal adenocarcinomas of low- to high-grade and the corresponding normal lymphocytes. The informative samples showed 12 out of 33 or about 36.4% LOH of this gene based on allelic loss of one or more polymorphic sites within the LOT1 genomic sequences. Similarly, primary breast carcinomas, which included invasive ductal (majority), spindle cell, mucinous, giant cell, and atypical medullary carcinomas, were examined on genomic DNA from patients for the allelic loss of LOT1. The informative cases showed 4 out of 10 samples or 40% LOH of this candidate tumor suppressor gene locus in breast cancer. CONCLUSIONS: The altered expression and LOH of the LOT1 locus support the gene's potential role, at least in part, in the pathogenesis of ovarian cancer and possibly in other types of cancer.

Alleles↗
PubMed2002

In situ analysis of the variable heavy chain gene of an IgM/IgG-expressing follicular lymphoma: evidence for interfollicular trafficking of tumor cells.

It is generally assumed that follicular lymphomas (FL) not only morphologically resemble normal germinal centers but have retained some functional characteristics of their non-neoplastic counterparts as well. Recent IgV gene analyses on a panel of FLs however, strongly suggested that FLs do not retain the capacity of somatic hypermutation and are not being selected on basis of the quality of their mIgV regions. To extend these findings, we investigated the follicular organization and class switching in a FL that consisted of both IgM- and IgG-expressing tumor cells with a high somatic mutation load and significant intraclonal V(H) gene diversity. V(H)-C(mu) and V(H)-Cgamma gene transcripts were amplified and sequenced from samples of approximately 50 tumor cells, isolated from frozen tissue sections by laser microdissection. We identified many different subclones and obtained limited evidence of subclone dominance in individual follicles. Remarkably, several subclones were found scattered over different follicles. All samples contained IgM- and IgG-expressing tumor cells with, in general, non-identical mutation patterns, which is not in support of ongoing class switching. Accordingly, no switch circle recombination products were found. The findings indicate that the neoplastic follicles lack the organization and functions typical of reactive germinal centers.

Cell Movement↗
PubMed2003

Clostridium difficile toxin B is an inflammatory enterotoxin in human intestine.

BACKGROUND & AIMS: Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis, diseases afflicting millions of people each year. Although C. difficile releases 2 structurally similar exotoxins, toxin A and toxin B, animal experiments suggest that only toxin A mediates diarrhea and enterocolitis. However, toxin A-negative/toxin B-positive strains of C. difficile recently were isolated from patients with antibiotic-associated diarrhea and colitis, indicating that toxin B also may be pathogenic in humans. METHODS: Here we used subcutaneously transplanted human intestinal xenografts in immunodeficient mice to generate a chimeric animal model for C. difficile toxin-induced pathology of human intestine. RESULTS: We found that intraluminal toxin B, like equivalent concentrations of toxin A, induced intestinal epithelial cell damage, increased mucosal permeability, stimulated interleukin (IL)-8 synthesis, and caused an acute inflammatory response characterized by neutrophil recruitment and tissue damage. Laser capture microdissection and real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) showed that intestinal epithelial cell-specific IL-8 gene expression also was increased significantly after luminal exposure to C. difficile toxins in vivo. CONCLUSIONS: We conclude that C. difficile toxin B, like toxin A, is a potent inflammatory enterotoxin for human intestine. Future therapeutic or vaccine strategies for C. difficile infection therefore need to target both toxins.

Adolescent↗
PubMed1996

Very early complete hydatidiform mole.

Recent trends toward early pregnancy ultrasound have led to evacuation of complete hydatidiform moles at a stage before the development of diffuse trophoblast hyperplasia and villous cavitation. Absence of these recognized diagnostic criteria can lead to misdiagnosis and subsequent trophoblastic neoplasia. The authors identified a case of very early complete hydatidiform mole (VECM) on review of a previous curettage specimen when the patient presented 4 weeks later with increasing human chorionic gonadotropin (HCC) titers and the typical histological features of complete mole on a subsequent curettage. DNA studies on this index case and three subsequent similar specimens confirmed the diagnosis of complete hydatidiform mole using polymerase chain reaction (PCR) amplification of eight microsatellite markers on microdissected maternal and villous tissue. VECM were compared with spontaneous abortions and elective terminations of a similar gestational age to develop diagnostic criteria. Five cardinal diagnostic features were identified: redundant bulbous terminal villi, hypercellular villous stroma, a labyrinthine network of vinous stromal canaliculi, focal cytotrophoblast and syncytiotrophoblast hyperplasia on both villi and the undersurface of the chorionic plate, and enlarged hyperchromatic implantation site trophoblast.

Adult↗
PubMed1999

Comparative microsatellite analysis in discerning origin of disseminated tumor: the case of a patient with malignant ascites and a history of multiple tumors.

The origin of metastatic carcinoma is now always easily resolved on the basis of conventional dinical and pathological parameters, particularly in patients with more than 1 primary tumor. When 1 of the tumors is a renal cell carcinoma, the clinical picture is further confounded by the tendency of these tumors to be locally silent, to metastasize to unusual sites, and to disseminate long after removal of the primary tumor. We compared tumors for loss (ie, deletion) of loci on chromosomal arms 3p, 5q, 11q, and 18q in a patient with a malignant ascites fluid, a remote history of renal and colonic neoplasms, and a strong clinical suspicion of disseminated gastrointestinal adenocarcinoma. DNA from microdissected tumors and normal tissues was subjected to polymerase chain reaction-based microsatellite analysis. Even though the clinical picture suggested a gastrointestinal origin, comparison of genetic alterations clearly showed that the malignant ascites represented recurrence of the renal cell carcinoma. The malignant ascites and the primary renal cell carcinoma showed identical patterns of allelic loss at all loci tested. In contrast, the malignant ascites and colonic adenoma showed discordant patterns of allelic loss. Comparative microsatellite analysis provides a rapid genetic approach for discerning the origin of metastatic tumor spread. This may be a useful diagnostic adjunct when tumor origin is not clear on clinical or morphological grounds. In some instances, it may even provide a reasonable alternative to an extensive and costly conventional work-up.

Adenoma↗
PubMed2003

Region-specific changes in NMDA receptor mRNA induced by chronic morphine treatment are prevented by the co-administration of the competitive NMDA receptor antagonist LY274614.

The steady-state mRNA levels of the NMDA receptor NR1 subunit were determined by a quantitative solution hybridization assay in selected CNS regions associated with antinociception in the rat. Tissues were obtained by microdissection from rats treated chronically with morphine alone or in combination with LY274614, a competitive NMDA receptor antagonist. Morphine treatment for 7 days resulted in the development of tolerance to morphine's analgesic effect and produced a significant decrease in the steady-state NR1 mRNA levels in the spinal cord dorsal horn (by 16%), and an elevation in nucleus raphe magnus and medial thalamus (by 26 and 38%, respectively). The NR1 mRNA levels were unchanged in the lateral paragigantocellular nucleus, locus coeruleus, periaqueductal grey, and sensorimotor cortex. NMDA receptor binding in the spinal cord measured with [3H]MK-801 was reduced approximately 50% by chronic morphine treatment. Co-administration of LY274614 (s.c. at 24 mg/kg/24 h via an osmotic pump) not only attenuated the development of morphine tolerance but also prevented the changes in the NR1 mRNA levels induced by chronic morphine administration. Neither a 7-day infusion of LY274614 nor an acute injection of morphine (10 mg/kg, s.c.) changed the NR1 mRNA levels. These results suggest that changes in the expression of the NR1 mRNA induced by chronic morphine in three CNS regions involved in antinociception are associated with the development of morphine tolerance and in the spinal cord, morphine tolerance is associated with the downregulation of NMDA receptors.

Animals↗
PubMed1999

LOH at the APC/MCC gene (5Q21) is frequent in early stages of non-small cell lung cancer.

Lung cancer is the leading cause of death in both women and men in the United States and many European countries. Molecular cytogenetic and LOH analyses of non-small cell lung cancer have shown somatic genetic alterations in a variety of chromosomes, such as 1p, 3p, 5q, 8p, 9p, 11p, 11q and 17p. Allelic deletions of the known tumor suppressor gene APC at 5q21 are frequently observed in advanced stages of lung cancer and have been correlated with poor prognosis in previous reports. We investigated 33 cases of NSCL for LOH at 5q21: 22 squamous cell and 11 adenocarcinomas. Normal and tumor cells were microdissected from paraffin embedded tissues and PCR amplification was performed utilising the specific markers D5S299 and D5S346 at 5q21 and PYGM at 11q13, respectively. Clinicopathological data, survival and recurrence rates were obtained in all cases. We detected LOH at 5q21 in 4/9 (44%) informative adenocarcinomas and in 13/16 (81%) informative SCC. LOH was frequent in early stages (12/15 stage I cases) and did not correlate with recurrence or poor survival. Our results show that LOH at 5q21 is more frequent in squamous cell carcinomas than in adenocarcinomas, is frequent in early stages of the disease, and does not have prognostic significance.

Adenocarcinoma↗
PubMed2003

Accurate and reproducible gene expression profiles from laser capture microdissection, transcript amplification, and high density oligonucleotide microarray analysis.

Gene expression profiling using high density oligonucleotide arrays is a powerful method to generate an unbiased survey of a cell's transcriptional landscape. Increasingly complex biological questions require that this approach be applicable to the small numbers of cells that are obtained from sources such as laser capture microdissection (LCM) of solid tissues. In this report, we demonstrate that two rounds of transcript amplification can generate accurate and reproducible gene expression profiles using high density oligonucleotide microarrays, starting with as little as 10 ng of total RNA. Biased amplification of the 3' end of transcripts does not have a major impact on the overall transcript profile due to the 3' bias of probe sets incorporated in the array design. Furthermore, greater than 95% of all genes detected demonstrate less than a twofold difference in expression when independent tissue dissections of identical cell populations are compared. The accuracy and technical reproducibility of the method suggests that expression profiling using transcript amplification and high density oligonucleotide microarrays can be used on a routine basis.

Breast Neoplasms↗
PubMed2005

Sporadic amyotrophic lateral sclerosis: a hypothesis of persistent (non-lytic) enteroviral infection.

Because of recently reported reverse transcriptase polymerase chain reaction evidence of enterovirus in sporadic amyotrophic lateral sclerosis (SALS) and because of newly available anti-enteroviral drugs binding enteroviral capsids, it is reasonable to re-formulate an enteroviral hypothesis of SALS using recent advances in molecular virology. Viral persistence is non-lytic and non-cytopathic infection that evades host's immune surveillance. Enteroviruses are known to cause persistent as well as lytic infection both in vitro and in vivo. Both virion as well as host factors modulate between persistent and lytic infection. Apoptosis, or programmed cell death, is a process of active non-necrotic cell death. It has complex interplay with viruses and may be either promoted or opposed by them. Apoptosis is a major factor in motor neuron death in SALS. Viral tropism is the process by which viruses select and propagate to target cells. It is controlled by capsid conformation and surface receptors on host cells. Enteroviruses have a region on their capsids known as the canyon which docks on such receptors. Docking induces conformational changes of the capsid and genome release. Poliovirus, tropic for motor neurons, docks on the poliovirus receptor, about which much is known. The virus penetrates the motor system focally after crossing either the blood-muscle or the blood-brain barriers. It propagates bidirectionally along axons and synapses to contiguous motor neurons, upper as well as lower, which sequester infection and create avenues for spread over long distances. If chronic and persistent rather than acute and lytic, such viruses trafficking in a finite system of non-dividing cells and inducing apoptosis would cause cell death that summates linearly rather than exponentially. Taken together, these explain signature clinical features of SALS - focal onset weakness, contiguous or regional spread of weakness, confinement to upper and lower motor neurons, and linear rates of progression. The hypothesis predicts the following testable investigations: 1) viral detection may be possible by applying amplification technology to optimally acquired nervous tissue processed by laser microdissection; 2) genetic susceptibility factors such as cell surface receptor polymorphisms may combine with sporadic exposure and chance penetration of the motor system in SALS; 3) a transgenic animal model might be created by inserting such genetic factors into an animal host and inoculating intramuscularly rather than intracerebrally biochemical fractions of SALS motor neurons at vulnerable periods in the developmental life cycle of the transgenic host; and 4) continual long-term administration of anti-enteroviral agents called capsid-binding compounds which stabilize capsids and prevent genome release might be efficacious.

Amyotrophic Lateral Sclerosis↗
PubMed1998

Distinguishing second primary tumors from lung metastases in patients with head and neck squamous cell carcinoma.

BACKGROUND: In patients with head and neck squamous cell carcinoma (HNSCC), a squamous cell carcinoma (SCC) in the lung represents either another primary tumor or a metastasis. This distinction greatly influences patient prognosis and could guide treatment strategies, but the nature of a solitary lung nodule is often difficult to discern by use of standard clinical and histologic parameters. Comparison of genetic alterations in the tumors could resolve this dilemma. METHODS: We compared paired tumors from 16 patients with HNSCC and a solitary lung SCC for loss (i.e., deletion) of loci on chromosomal arms 3p and 9p. Losses at these loci occur early during neoplastic transformation of the respiratory tract. DNA from microdissected tumors and normal tissues was subjected to polymerase chain reaction-based microsatellite analysis. An effort was also made to distinguish primary lung cancers from lung metastases on the basis of clinical and histopathologic features. RESULTS: In most cases, comparison of genetic alterations clarified the relationship between the lung tumor and the primary HNSCC. The paired tumors from 10 patients had concordant patterns of loss at all loci suggesting metastatic spread, whereas three paired tumors had discordant patterns of loss at all loci suggesting independent tumor origin. These observations were supported by the clinical and pathologic findings. CONCLUSIONS/IMPLICATIONS: In patients with HNSCC and a solitary SCC in the lung, microsatellite analysis provides a rapid genetic approach for discerning clonal relationships. In such patients, we found that a solitary SCC in the lung more likely represents a metastasis than an independent lung cancer. Microsatellite analysis could potentially be applied to any patient with multiple tumors, where tumor relationships are not clear on clinical, radiographic, or even histopathologic grounds.

Adult↗
PubMed1997

COBRA: a sensitive and quantitative DNA methylation assay.

We report here on a quantitative technique called COBRA to determine DNA methylation levels at specific gene loci in small amounts of genomic DNA. Restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA as described previously. We show that methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA methylation levels. In addition, we show that this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples. COBRA thus combines the powerful features of ease of use, quantitative accuracy, and compatibility with paraffin sections.

Base Sequence↗