Optimization of acoustic coupling for bottom actuated scattering based subsurface scanning probe microscopy.

The characterization of buried nanoscale structures nondestructively is an important challenge in a number of applications, such as defect detection and metrology in the semiconductor industry. A promising technique is Subsurface Scanning Probe Microscopy (SSPM), which combines ultrasound with Atomic Force Microscopy (AFM). Initially, SSPM was used to measure the viscoelastic contrast between a subsurface feature and its surrounding medium. However, by increasing the ultrasonic frequency to >1 GHz, it has been shown that SSPM can also measure acoustic impedance based contrasts. At these frequencies, it becomes difficult to reliably couple the sound into the sample such that the AFM is able to pick up the scattered sound field. The cause is the existence of strong acoustic resonances in the sample, the transducer, and the coupling layer-the liquid layer used to couple the sound energy from the transducer into the sample-in combination with the nonlinearity of the tip-sample interaction. Thus, it is essential to control and measure the thickness of the coupling layer with nanometer accuracy. Here, we present the design of a mechanical clamp to ensure a stable acoustic coupling. Moreover, an acoustic method is presented to measure the coupling layer thickness in real-time. Stable coupling layers with thicknesses of 700 ± 2 nm were achieved over periods of 2-4 h. Measurements of the downmixed AFM signals showed stable signal intensities for >1 h. The clamp and monitoring method introduced here makes scattering based SSPM practical, robust, and reliable and enables measurement periods of hours.

Nucleolar changes during the first steps of experimental hepatocarcinogenesis in rats.

Silver stainability of hepatocytes as an expression of nucleolar activity was studied in vivo during rat hepatocarcinogenesis. Male Wistar rats were injected with one dose of diethylnitrosamine (200 mg/kg body weight dissolved in 0.9% NaCl), followed by a selection procedure with a short exposure to 2-acetylaminofluorene in combination with a proliferative stimulus, such as the administration of CCl4. Finally, after 1 week of a normal diet, some of the rats were treated with phenobarbital. After enzymatic isolation, the hepatocytes were silver stained; the estimation of nucleolar activity was determined by a cytomorphologic analysis of the silver-stained nuclei. It was demonstrated that during the first steps of hepatocarcinogenesis, both diethylnitrosamine, as initiator, and phenobarbital, as promotor, induce modifications of the nucleolar morphology in silver-stained hepatocytes.

Different competition of thyroxine binding to transthyretin and thyroxine-binding globulin by hydroxy-PCBs, PCDDs and PCDFs.

In an earlier study several hydroxylated polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) competitively displaced [125I]thyroxine (T4) from transthyretin with different potencies. Transthyretin is the major T4 transport protein in plasma of rodents. In man, however, thyroxine-binding globulin transports most of the T4 in blood. In this study, hydroxylated PCBs, PCDDs and PCDFs were tested in an in vitro competitive binding assay, using purified human thyroxine-binding globulin and [125I]T4 as the displaceable radioligand. None of the tested hydroxylated PCBs, PCDDs and PCDFs inhibited [125I]T4 binding to thyroxine-binding globulin. In addition, some T4 derived compounds, e.g., tyrosine, mono-iodotyrosine, di-iodotyrosine and tri-iodophenol were tested on both transthyretin and thyroxine-binding globulin to investigate possible differences in structural characteristics determining T4 binding to thyroxine-binding globulin and transthyretin. The T4 derived compounds also did not inhibit [125I]T4 binding to thyroxine-binding globulin as tested in the in vitro assay. However, tri-iodophenol and to a lesser extent di-iodotyrosine inhibited [125I]T4-transthyretin binding. These results indicate a marked difference in T4 binding to thyroxine-binding globulin or transthyretin. The hydroxylated PCBs, PCDDs and PCDFs can inhibit T4 binding to transthyretin, but not to thyroxine-binding globulin, and thus may cause different effects in rodents and man.

Serum alkaline DNase activity in normal or nonhospitalised individuals.

According to previous observations, the variations in serum alkaline DNase activity (SADA) appeared to be useful in monitoring malignant disease. In this study, SADA was measured in 625 individuals to explore nontumor-related factors which may influence SADA levels. The overall range in SADA was 0.2-82.3 kU/l. Women aged 50-79 years had higher (p less than 0.001) levels of SADA than younger females. A similar but less consistent effect of age was noticed in men (0.01 less than p less than 0.05). Older men had lower (0.01 less than p less than 0.05) SADA levels than the older women. Old women substituted with estrogens had lower (0.01 less than p less than 0.05) levels of SADA than those not treated with estrogens. SADA levels in pregnancy as well as postparturition were lower (p less than 0.001) than SADA values in nonpregnant females of similar age. In fertile women, no SADA variation was observed during the menstrual cycle and there was no significant effect of contraceptive pills. In males, SADA seemed unrelated to testosterone or cortisol levels but varied during the day. Smoking, alcohol consumption and drug therapy appeared to be without effect on SADA.

Separate isolation of cells from nodules and surrounding parenchyma of the same precancerous rat liver: biochemical and cytochemical characterization.

Various enzyme and metabolic alterations have been observed in the hyperplastic nodules which appear during the hepatocarcinogenesis. These alterations have been mainly specified by histochemical observations. In this report, a technique of hepatocyte isolation is described which enables the separation of 2 cellular fractions, respectively, from the nodules and from the surrounding parenchyma of the same liver of a rat previously treated with a hepatocarcinogen. Such a technique allowed parallel analysis of both cellular populations by biochemical and cytochemical techniques.

Variations in serum alkaline DNase activity: a possible clinical test for therapeutic prognosis of human tumors.

Previously published histochemical observations indicated that variations in serum alkaline DNase activity (SADA) could be considered as a possible prognostic test for human tumor therapy. In more than 80 cancer patients biochemical measurements of SADA were performed using the spectrophotometrical technique. A decrease of SADA promptly after the beginning of tumor treatment (phase I) may be interpreted as an early sign of therapeutically induced tumor necrosis and as a positive response to the treatment. A delayed regain of SADA (phase II) can predict the long term evolution of the disease. In this phase (II), a regain of SADA up to values higher than the initial value corresponds to a complete tumor regression. If the regain is limited to values lower than the initial value, only a partial tumor regression is seen. No variations of SADA were observed in patients without therapeutic response and with fatal evolution.

Structure-dependent, competitive interaction of hydroxy-polychlorobiphenyls, -dibenzo-p-dioxins and -dibenzofurans with human transthyretin.

Previous results from our laboratory indicated specific and competitive interactions of hydroxylated metabolites of 3,3', 4,4'-tetrachlorobiphenyl with the plasma thyroid hormone transport protein, transthyretin (TTR), in rats in vivo and with human TTR in vitro. In the present study the structural requirements for competition with thyroxine (T4) for TTR-binding were investigated in more detail. Several hydroxylated polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) were tested in an in vitro competitive binding assay, using purified human TTR and [125I]T4 as a displaceable radioligand. All hydroxylated PCBs, but not the single PCB tested, competitively displaced [125I]T4 from TTR with differential potency. The highest competitive binding potency was observed for hydroxylated PCB congeners with the hydroxygroup substituted on meta or para positions and one or more chlorine atoms substituted adjacent to the hydroxy group on either or both aromatic rings (IC50 range 6.5-25 nM; Ka range: 0.78-3.95 x 10(8) M-1). The relative potency of all meta or para hydroxylated PCBs was higher than that of the physiological ligand, T4 (relative potency range: 3.5-13.6 compared to T4). There were no marked distinctions in TTR-T4 competitive binding potencies between the ortho- and non-ortho-chlorine substituted hydroxy-PCB congeners tested. Marked differences in TTR-T4 binding competition potency were observed between the limited number of hydroxylated PCDDs and PCDFs tested. The hydroxy-PCDD/Fs, with chlorine substitution adjacent to the hydroxy-group, i.e. 7-OH-2,3,8-trichlorodibenzo-p-dioxin, 2-OH-1,3,7,8-tetrachlorodibenzo-p-dioxin and 3-OH-2,6,7,8-tetrachlorodibenzofuran, all showed a similar or higher relative binding potency, i.e. 1, 4.4 and 4.5 times higher, respectively, than T4. No detectable [125I]T4 displacement was observed with 2-OH-7,8-dichlorodibenzofuran, 8-OH-2,3,4-trichlorodibenzofuran and 8-OH-2,3-dichlorodibenzo-p-dioxin, which did not contain chlorine substitution adjacent to the OH-group. These results indicate a profound similarity in structural requirements for TTR binding between hydroxy-PCB, -PCDD and -PCDF metabolites and the physiological ligand, T4, e.g. halogen substitution adjacent to the para hydroxy group, while planarity does not seem to influence the ligand-binding potency.

Use of monolayers of primary rat kidney cortex cells for nephrotoxicity studies.

Kidney cells were isolated from rat kidney cortex and maintained in short-term monolayer cultures. A number of important parameters were studied in order to establish the usefulness of these cells for toxicity studies. Despite morphological differences between the cultured cells and similar cells in vivo, many relevant enzyme systems remained present and functional. Intracellular glutathione levels were stable up to 5 days in culture. The glutathione S-transferase activity during culture remained stable although at a lower level than in freshly isolated cells. Whereas rat kidney cytosol contained subunits 4, 7, 2 and 1, 3- and 5-day-old cultures contained glutathione transferase subunits 7, 2 and a small amount of subunit 1. Cytochrome P-450, although measurable in microsomes from freshly isolated cells, could not be determined after 1 day in culture. Organic anion transporters on the basolateral side and gamma-glutamyl transpeptidase on the apical side were present. Through cytotoxicity studies, beta-lyase activity could be demonstrated in the culture. Hence this monolayer culture system, which can be used in combination with filters, seems to be suitable for studying various mechanisms of nephrotoxicity.

[Possible value of variations in the serum alkaline DNAse activity in the prognosis of cancers of the upper respiratory-digestive tract treated by chemotherapy]. / Valeur prédictive des variations de l'activité sérique de la DNAse alcaline dans le pronostic des cancers des voies aérodigestives supérieures traités par chimiothérapie.

Variations in serum alkaline DNase activity before and repeatedly after standardized chemotherapy were examined in patients with head and neck carcinomas. The enzyme activity was measured by way of a modified spectrophotometric method. No variations of such activity observed in patients without therapeutic response or with minor response could be considered as a marker of primary or acquired resistance to chemotherapy. Distinct variations in serum alkaline DNase activity (a steep decrease after therapy followed a few weeks later by a regain of values higher than the initial value) correspond to complete or partial positive responses. Such observations of the variations in enzyme activity in relation to individual initial values measured before therapy could be considered as a reliable prognostic test for the therapy of many head and neck carcinomas.

Effect of antiretroviral treatment and counselling on disclosure of HIV-serostatus in Johannesburg, South Africa.

This prospective non-randomized study of clinic attendees, compares self-reported HIV disclosure patterns in relation to access to antiretroviral access and counselling. It was carried out in public sector hospital HIV clinics in Johannesburg, South Africa, and 144 HIV-positive men and women attending the HIV clinics participated in the study.The results showed that there was no correlation between being on antiretroviral therapy and disclosure of HIV status. There was also no correlation between disclosure of HIV status and with different levels of counselling and access to support groups. Disclosure levels were high (92% told at least one person), however, there was a high level of delayed (15% greater than a year) or non-disclosure (21%) to partners. Family members and partners provided most moral support after disclosure. Having access to antiretroviral therapy and support groups and available counselling did not seem to affect disclosure patterns. It is possible that a patients beliefs about their treatment plays a more important role for disclosure than the actual treatment itself. Other factors are also likely important for disclosure, such as the patient's social network especially with their families, and knowledge of the disease.

Promoting effect of oxazepam in rat hepatocarcinogenesis.

In order to check whether the benzodiazepine, oxazepam (OZ), has a modulating effect on the development of liver cancer, it was given to rats previously submitted to two different protocols of hepatocarcinogenesis: the resistant hepatocyte protocol and Pitot's model (initiation-promotion). Its effects were compared with those of phenobarbital (PB) administered under the same conditions. As compared with basal diet, a diet containing 0.05% of PB and 0.1% of OZ enhanced, in both models, the development of gamma-glutamyltransferase-positive lesions in early stages. OZ also had a promoting effect on the development of liver cancers in later stages in both models whereas PB only enhanced it in the resistant hepatocyte protocol. Thus, like PB, OZ may have a promoting effect on liver cancer in rats.

Dietary modulation of rat liver carcinogenesis.

Rat liver carcinogenesis was induced according to the resistant hepatocyte model of Solt and Farber. One week after the end of the procedure for the rapid growth of altered hepatocytes, one group of rats was submitted to a high fat (20%) regimen up to the end of the experiment. The incidence of histologically confirmed malignant hepatocarcinomas was compared with that observed in a group that remained on a normal diet. The modulating (promoting) effect of the high fat regimen was evident since nine out of 10 animals in this group bore macroscopically detectable tumors and eight out of 10 presented histologically confirmed hepatocellular carcinomas as early as 24 weeks after the beginning of the experiment. At that time, no malignant tumors were detected in the group submitted to the normal fat regimen. These results are similar to those resulting from a porto-caval shunt or the chronic administration of liver tumor promoters. This suggests that at this stage of the carcinogenic process, any treatment inducing chronically metabolic adaptation in a tissue containing preneoplastic nodules modulates positively the progression of these lesions as demonstrated by the dramatic reduction of the lag period for their malignant transformation.

Non-toxic potentiation of cancer chemotherapy by combined C and K3 vitamin pre-treatment.

The influence on the survival of ascitic liver tumor (TLT)-bearing mice of combined vitamins C and K3 administered before or after a single i.p. dose of 6 different cytotoxic drugs, all commonly used in human cancer therapy, was investigated. Combined i.p. administration of these vitamins produced a distinct chemotherapy-potentiating effect for all drugs examined, especially when injected before chemotherapy. This potentiating treatment did not increase the general and organ toxicity that accompanies cancer chemotherapy. The possible generation of peroxides followed by membrane lipid alteration, DNase activation and DNA destruction by combined vitamin C and K3 in catalase-deficient cancer cells might be involved in the mechanisms of this selective potentiation.

Comparison of the biological effects of phenobarbital and nafenopin on rat hepatocarcinogenesis.

In order to further analyze the biological effects of phenobarbital (PB) and nafenopin (NAF) on rat hepatocarcinogenesis, four experiments were undertaken. In the first one, their "promoting" effect on an ongoing carcinogenic process was analyzed. Rats were initiated by diethylnitrosamine treatment (I) and submitted two weeks later to a selection procedure (S). One week after 2-acetylaminofluorene (2-AAF) release, the animals received for up to 56 weeks a basal diet or a diet containing 0.05% of PB or 0.1% of NAF. The quantitative analysis of the gamma-glutamyl-transferase-positive lesions showed that, 8 to 19 weeks after I, PB enhanced the development of preneoplastic lesions whereas NAF inhibited it as compared to a group receiving a basal diet. However, both compounds enhanced the incidence and the yield of liver cancer starting 27 weeks after I (67% and 95%, respectively, vs 10%). In the second experiment, the effect of chronic administration of PB and NAF given after I without S or after S without I was analyzed. Within a period of observation of 27 to 32 weeks, the incidence of cancer was 10% after I/PB and 75% after I/NAF. No cancer developed after S/PB, S/NAF or NAF alone. The third experiment was designed to test whether NAF had an initiating or selecting effect. The results of the quantitative analysis of the gamma-glutamyl-transferase-positive lesions showed that as compared to diethylnitrosamine, NAF had no initiating effect. When NAF replaced 2-AAF in the selection procedure, few gamma-glutamyl-transferase-positive lesions and no cancer were detected 8 and 32 weeks after I. The fourth experiment indicated that NAF could not prevent the remodeling of preneoplastic lesions induced in the I/S protocol. Even though they both have a "promoting" effect in liver carcinogenesis as evidenced by the increased incidence and yield of cancer, PB and NAF act differently.

Influence of the nature and the dose of the initiator on the development of premalignant and malignant lesions in rat hepatocarcinogenesis.

Using a triphasic protocol recently described to induce malignant tumors in rat liver, the question has been asked whether both the nature and the dose of the initiator influence the carcinogenic process. Two nitrosamines (diethylnitrosamine DEN and N-nitrosomorpholine NNM) have been used to initiate that process. With regard to the premalignant stages appearing in the liver up to 19 weeks after initiation, there is a dose-dependent relationship between the dose of initiator and both the percentage of the parenchyma occupied by and the number of GGT+ lesions. DEN is always more potent than equivalent doses of NNM. With regard to malignant tumors appearing within the period of observation (up to 44 weeks), the two highest doses (100 and 200 mg/kg) of DEN appear to be the only carcinogenic treatments. Cancer incidence (percentage of rats bearing histologically characterized malignant tumor) is the same after both treatments, but the tumor yield (number of tumors per rat bearing macroscopic tumors) is higher (+/- 2X) after 200 mg/kg than after 100 mg/kg.

Comparative analysis of the effect of phenobarbital, dichlorodiphenyltrichloroethane, butylated hydroxytoluene and nafenopin on rat hepatocarcinogenesis.

In order to investigate whether different 'promoters' have the same qualitative and/or quantitative effects on rat hepatocarcinogenesis, 0.05% of phenobarbital (PB), 0.05% of dichlorodiphenyltrichloroethane (DDT), 0.5% butylated hydroxytoluene (BHT) and 0.1% of nafenopin (NAF) were chronically administered in the diet to rats previously submitted to an initiation by diethylnitrosamine and a selection with 2-acetylaminofluorene plus CC14. The animals were killed after 3, 6 and 14 weeks of 'promoters' administration to analyse their effect on premalignant lesions. The quantitative analysis of the gamma-glutamyltransferase positive lesions indicates that as compared to a control group receiving a basal diet after initiation and selection, PB, DDT and BHT enhance the development of these lesions whereas NAF inhibits it. Rats were also killed after 22 weeks of administration to analyse the incidence and the yield of liver cancer. As compared to the control group, PB, DDT and surprisingly NAF enhance the development of liver cancer whereas BHT does not. This suggests that the effect of potential 'promoters' should be analysed on cancer development rather than on premalignant lesions.

Serum alkaline deoxyribonuclease activity, a sensitive marker for the therapeutic monitoring of cancer patients: methodological aspects.

Alkaline deoxyribonuclease (DNase) is present in human circulating serum but its physiological role and signification of its variations are still largely unknown. The present report demonstrates that between 37 degrees C and 50 degrees C, as measured in the presence of 0.25 mmol/l Ca2+ and 5 mmol/l Mg2+, serum alkaline DNase activity increases, in most sera, reaching a level far higher than expected from thermal activation. This observation is thought to be due to the thermal inactivation of a serum inhibitor of the enzyme, which limits its usefulness as a therapeutic marker. By measuring serum alkaline DNase activity at 50 degrees C, the authors have developed a clinical test which has been successfully applied to the therapeutic monitoring of patients with various types of cancers.

Variations in serum alkaline DNase activity in rats during growth and treatment of tumors sensitive or resistant to therapy.

Serum alkaline DNase activity (SADA) was investigated in rats receiving s.c. transplants of tumor cells sensitive or resistant to chemotherapy. Serum samples from each animal were collected before transplantation, during the development of tumors and after therapy. Within a few days after transplantation of both tumor lines (sensitive or resistant), SADA levels decreased progressively to 52% of the normal pre-transplantation level (p less than 0.01). This decrease in SADA preceded by 4 to 5 days the appearance of any palpable tumor mass. In all untreated animals as well as in treated rats bearing resistant tumors, SADA remained at a low level until death. In rats bearing tumors sensitive to therapy a progressive increase in SADA was observed after treatment, paralleling tumor regression. When tumor regression was complete, SADA resumed the levels of activity measured prior to transplantation.

Histochemical detection of the in vivo produced cellular aldehydes by means of direct Schiff's reaction in CCl4 intoxicated rat liver.

A histochemical technique for detection of the in vivo induced cellular aldehydes based on the direct Schiff's reaction is reported in this paper. CCl4-intoxicated rat liver was used as an experimental model. Fresh and non-pretreated rat liver cryostat sections fixed in 10% formol calcium solution and washed in distilled water were exposed to Schiff's reagent. The sections were then immersed in two baths of sodium bisulphite solution, then in water, dehydrated in ethanol, cleared in xylene and mounted in a synthetic anhydrous mounting medium. As Schiff positive areas presented well circumscribed foci which increased with time following intoxication, semi-quantitative planimetric measurements were feasible. The direct Schiff's reaction detects cellular aldehydes in a sensitive, rapid, histologically and topographically estimable way. The appearance of these aldehydes precedes distinctly morphological alterations detectable by other histochemical or histological techniques. No positive results were obtained in control, non-intoxicated rat livers. Inhibition of this direct Schiff's reaction was obtained in positive control rat liver sections preincubated in solutions of aldehyde blockers. Histochemical detection of aldehydes may give useful information on different aspects of tissue and organ intoxication such as their topography, appearance, evolution, extension, consequences and effects of treatment. The direct Schiff's reaction can be considered as a valuable tool in fundamental and applied research dealing with various toxicological, environmental, pathological, cancer-related and therapeutic problems.