Applications of DNA Flow Cytometry and Fluorescence In situ Hybridization Using a Chromosome-specific DNA Probe on Paraffin-embedded Tissue Sections of Primary Malignant Melanomas

We have applied DNA flow cytometric analysis to paraffin-embedded tissue sections of primary malignant melanomas. Conventionally, flow cytometric analysis of paraffin-embedded tissue sections has been done by the method of Hedley et al. We added ultrasound treatment to the method of Hedley et al. and a lower value of coefficient of variation was shown. Furthermore, a new technique, fluorescence in situ hybridization with a chromosome-specific repetitive DNA probe, was used for the analysis of chromosomal numerical aberrations in the same paraffin-embedded tissue sections. The DNA flow cytometric analysis showed that in 8 cases six primary malignant melanomas were of the aneuploid pattern and two cases of lentigo maligna (melamona in situ) were of the diploid pattern. By fluorescence in situ hybridization, the two cases with the diploid pattern had spots/nucleus of 1.28 and 1.12, and those with the aneuploid pattern had spots/nucleus from 2.01 to 2.27. Only one nodular melanoma in an aneuploid case showed spots/nucleus of 1.71. These data indicate that fluorescence in situ hybridization with chromosome-specific repetitive DNA probes can serve as a cytogenetic tool for the analysis of interphase nuclei of solid human tumors and may be useful for the study of tumor cell heterogeneity.     

In vivo receptor binding,neurochemical and functional studies with the dopamine D-1 receptor antagonist SCH 23390

Summary A series of in vivo experiments were undertaken, relating functional (motor activity, body temperature), dopamine (DA) receptor binding and neurochemical (catecholamine synthesis and utilization, DA release) aspects of the pharmacology of SCH 23390 in the rat.The compound inhibited the locomotor hyperactivity, but not the hypothermia, induced by the potent DA stimulant DP-5,6-ADTN. Interstingly, SCH 23390 simultaneously failed to displace DP-5,6-ADTN from its binding sites in the rat striatum—used as a direct in vivo biochemical index of DA (D-2) receptor interaction. The spontaneous locomotion in non-pretreated rats was likewise inhibited by SCH 23390. The locomotor-suppressive action, but not the DP-5,6-ADTN-displacing capcity of the D-2 blocker haloperidol was significantly enhanced by SCH 23390, suggesting that motility can be suppressed by either enhanced D-1 or D-2 (postsynaptic) receptor blockade, but also that the D-1 and D-2 sites involved may be physically distinct.SCH 23390 only slightly altered in vivo neurochemical of DA synthesis, release and nerve-impulse flow, indicating that, while similar in suppressing dopaminergic behaviour, the D-1 antagonist is less effective than traditional neuroleptics as an activator of DA neuronal feedback mechanisms. The weak increases of DA synthesis and release nonetheless obtained were equal in magnitude (30–40%) in the limbic vs. striatal brain areas; also in this respect, SCH 23390 thus differs from classical neuroleptics, which generally display more marked effects in the striatum than in limbic tissue.No major changes in the in vivo indices of NA synthesis and utilization (or in 5-HT synthesis) were found after SCH 23390 administration, by and large supporting the DA receptor specificity of the compound.In summary, the studies demonstrated that SCH 23390 can offset and accentuate, respectively, behavioural consequences of D-2 receptor stimulation and blockade. Importantly, at the same time no direct interaction at the level of D-2 DA receptor sites in the striatum was detected. Only slight, D-2 antagonist-like, changes in neurochemical indices of dopaminergic activity were observed after D-1 receptor blockade by means of SCH 23390. With regard to DA agonist hypothermia, SCH 23390 was without effect per se, but (at a high dose) attenuated the action of the D-2 antagonist haloperidol. The observations may indicate that the complex interactions between central D-1 and D-2 receptor-controlled mechanisms that influence behaviour, neurochemistry, and possibly autonomic nervous expression, are not identical.     

Nucleic acid spot hybridization with nonradioactive labeled probes in screening for human papillomavirus DNA sequences

We examined 161 human tissue samples using the spot hybridization technique with nonradioactive labeled DNA probes of human papillomavirus (HPV). Whole cells were spotted on nitrocellulose filters; DNA of the cells was denatured and fixed to the filter. Then the DNA spots were hybridized to nonradioactive labeled DNA and monitored by a sandwich immunoenzymatic reaction. This technique is simple, sensitive, specific, requires no special equipment, and can be used in clinical settings. HPV DNA was found in 92% of samples in which, on the basis of histologic and colposcopic criteria, its presence was suspected, as well as in 31 samples where it was not suspected.     

In vitro antibacterial activity of the volatile oil of Nigella sativa seeds against multiple drug-resistant isolates of Shigella spp. and isolates of Vibrio cholerae and Escherichia coli

The antibacterial activity of the volatile oil of Nigella sativa seeds was studied against 37 isolates of Shigella dysenteriae 1, Shigella flexneri, Shigella sonnei and Shigella boydii and 10 strains of Vibrio cholerae and Escherichia coli. Most of the strains were clinically resistant to ampicillin, co-trimoxazole and tetracycline. All the strains tested showed promising sensitivity to the volatile oil. The minimum inhibitory concentration (MIC) of the volatile oil for Shigella, Vibrio and Escherichia strains tested was between 50–400 μg/mL.     

Dorsal root ganglia cocultured with macrophages: an in vitro model to study experimental demyelination

The present investigation introduces an in vitro model to study macrophage properties during demyelination. Rat dorsal root ganglia (DRG) were cultured for obtaining myelinated peripheral nerve fibers. These cultures were exposed to non-resident macrophages. In untreated control cultures, there was no indication of myelin removal by the added macrophages. DRG were exposed to enzymatically generated oxygen radicals using the xanthin/xanthin oxidase or the glucose/glucose oxidase system. Assessment of Schwann cell viability and ultrastructural morphology revealed different patterns of cell cytotoxicity and morphological changes in different experiments. High concentrations caused complete tissue necrosis of the DRG, while low concentrations did not affect either cell viability or ultrastructural morphology. Under intermediate experimental conditions, oxygen radicals caused non-lethal Schwann cell damage leading to Schwann cell retraction and myelin sheath rejection. Myelin lamellae were disrupted and decompacted. These changes were followed by a selective macrophage attack on myelin sheats, resulting in demyelination. Axons, Schwann cells and sensory ganglion cells survived this attack. The specificity of the oxygen radical effects was tested in experiments using the oxygen radical scavengers catalase and superoxide dismutase. Catalase prevented the described effects on cell morphology and subsequently blocked demyelination by non-resident macrophages.Supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) (Br 1274/1-1)     

UROLITHIASIS IN AUSTRALIAN ABORIGINAL CHILDREN

Thirty-six Australian Aboriginal children with urolithiasis were reviewed. Males dominated the series. The age distribution ranged from 8 months to 12 years and nearly 70% were 2 years or younger. Thirty-five patients had upper tract stones. Ultrasound was diagnostic in 35 patients and was falsely negative in one. Dietary factors, dehydration and recurrent diarrhoea are incriminated in the aetiology, because ammonium urate and oxalate were the main constituents of the stones. Malformations of the urinary tract were rare and known metabolic disorders were not seen. Chemical dissolution of the stones was found to be a safe and effective adjuvant in the management of urate stones.     

Triploid origin of the gibel carp as revealed by 5S rDNA localization and chromosome painting

5S ribosomal DNA (rDNA) was isolated and sequenced from the gibel carp Carassius auratus gibelio with 162 chromosomes and crucian carp Carassius auratus with 100 chromosomes, and fluorescent probes for chromosome localization were prepared to ascertain the ploidy origin and evolutionary relationship between the two species. Using fluorescence in-situ hybridization (FISH), major 5S rDNA signals were localized to the short arms of three subtelocentric chromosomes in the gibel carp and to the short arms of two subtelocentrics in the crucian carp. In addition, some minor signals were detected on other chromosomes of both species. Simultaneously, six chromosomes were microdissected from the gibel carp metaphase spreads using glass needles, and the isolated chromosomes were amplified in vitro by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). Significantly, when the DOP-PCR-generated probes prepared from each single chromosome were hybridized, three same-sized chromosomes were painted in each gibel carp metaphase, whereas only two painted chromosomes were observed in each crucian carp metaphase spread. The data indicate that gibel carp is of triploid origin in comparison with diploid crucian carp.     

Erb:YAG and Hol:YAG laser ablation of meniscus and intervertebral discs.

Erb:YAG and Hol:YAG laser ablation rates of fibrocartilage and nucleus pulposus were measured in vitro simulating clinical conditions. After ablation macroscopic and microscopic appearance of the ablation site was investigated. Hol:YAG and Erb:YAG laser mean ablation rates increased almost linearly with rising energies, showing higher total ablation rates for the Hol:YAG laser due to its higher achievable energy density. At comparable energy densities the Erb:YAG laser appears to be more effective with respect to the corresponding ablation rates. Consequently, the ablational threshold proved to be lower for the Erb:YAG laser. Whereas during Hol:YAG laser ablation, some smoke formation and considerable tissue shrinking occurred, these effects could not be observed during Erb:YAG laser ablation. Consequently macroscopic and microscopic inspection showed some thermal damage after Hol:YAG and only minimal alterations after Erb:YAG laser ablation. Adjacent thermal damage was determined and proved to be lower for the Erb:YAG laser. In our opinion the characteristics of each laser system provide certain advantages for special clinical indications.     

Morphology and immunohistochemistry of carcinoma in situ adjacent to testicular germ cell tumours in adults and children: implications for histogenesis

Observations differ on the pre-invasive malignant lesions associated with the various categories of testicular germ cell tumours. Such lesions have been found to be similar in appearance and are assumed to be composed of multipotent cells, or conversely a distinctive pre-invasive stage has been reported in association with each form of germ cell neoplasm. This study was undertaken to see whether distinctive morphological and immunohistochemical features of carcinoma in situ adjacent to various categories of germ cell tumours could be established. Carcinoma in situ adjacent to seminomas, teratomas and mixed germ cell tumours in 18 adults was indistinguishable morphologically. Placental alkaline phosphatase was demonstrated immunohistochemically but vimentin and low molecular weight cytokeratins were uniformly absent in these abnormal germ cells from all three groups. These findings support the concept of a multipotent pre-invasive malignant cell for both seminoma and teratoma in the adult. Carcinoma in situ was not seen adjacent to 15 spermatocytic seminomas, nor was placental alkaline phosphatase demonstrated in tubules adjacent to these tumours. These negative findings are additional evidence that spermatocytic seminoma differs from classical seminoma in its histogenesis. Carcinoma in situ, as defined morphologically and immunohistochemically in adults, was not identified adjacent to yolk sac tumours and differentiated teratomas in 20 prepubertal testes. The possibility that pre-invasive malignancy in children may not resemble that in adults must be considered when assessing the malignant potential of cryptorchid testes on biopsies taken during orchidopexy.     

Effects of antisense mediated inhibition of cathepsin K on human osteoclasts obtained from peripheral blood.

Cathepsin K is a cystein protease that displays a proteolytic activity against Type I collagen and is abundantly and selectively expressed in osteoclasts where it plays a critical role in bone degradation. Its direct role in bone tissue has been defined by knock-out mice studies and inhibiting strategies in animals models. However, direct proof of cathepsin K function in human osteoclast model in vitro is lacking. The aim of this study is to analyze cathepsin K expression and localization in human osteoclasts obtained from peripheral blood and to examine cathepsin K function in these cells by antisense oligodeoxynucleotide (AS-ODN) strategy. AS-ODN was added to the culture of osteoclast precursors induced to differentiate by RANKL and M-CSF. AS-ODN treatment produced a significant down-regulation of cathepsin K mRNA (>80%) and protein expression, as verified respectively by Real-time PCR and by immunocytochemistry or Western blot. The cathepsin K inhibition caused an impairment of resorption activity as evaluated by a pit formation assay ( p = 0.045) and by electron microscopy, while the acidification process was unaffected. We demonstrated that antisense strategies against cathepsin K are selectively effective to inhibit resorption activity in human osteoclasts, like in animal models.