The role of testosterone in the feedback control of male FSH and LH secretion

Summary Intramuscular administration of 250 mg testosterone oenanthate per week over a period of 21 weeks treatment rapidly and sustainedly suppressed serum LH as well as FSH levels in seven normal males, while serum testosterone rose by a factor of approximately two. These together with other data provide increasing evidence for a feedback control of FSH secretion by gonadal steroids in the male in addition to the already described but as yet undefined tubular testicular factor.     

Creating a mission-based reporting system at an academic health center.

The authors developed a Web-based mission-based reporting (MBR) system for their university's (UC Davis's) health system to report faculty members' activities in research and creative work, clinical service, education, and community/university service. They developed the system over several years (1998-2001) in response to a perceived need to better define faculty members' productivity for faculty development, financial management, and program assessment. The goal was to create a measurement tool that could be used by department chairs to counsel faculty on their performances. The MBR system provides measures of effort for each of the university's four missions. Departments or the school can use the output to better define expenditures and allocations of resources. The system provides both a quantitative metric of times spent on various activities within each mission, and a qualitative metric for the effort expended. The authors report the process of developing the MBR system and making it applicable for both clinical and basic science departments, and the mixed success experienced in its implementation. The system appears to depict the activities of most faculty fairly accurately, and chairs of test departments have been generally enthusiastic. However, resistance to general implementation remains, chiefly due to concerns about reliability, validity, and time required for completing the report. The authors conclude that MBR can be useful but will require some streamlining and the elimination of other redundant reporting instruments. A well-defined purpose is required to motivate its use.     

Histone H1-mediated transfection: role of calcium in the cellular uptake and intracellular fate of H1-DNA complexes

Previously, we have shown that the transgene expression in the endothelial cell line ECV 304 strongly depends on the presence of low concentrations of Ca2+. However, it remained unclear, which transfection steps are controlled by Ca2+ ions. In the present study, we constructed transfection complexes of digoxigenin-labelled DNA and FITC-labelled histone H1. We monitored the pathway of these complexes with the use of anti-digoxigenin and anti-cathepsin B antibodies and immunofluorescence microscopy. Double labelling of DNA and cathepsin B permitted the localization of transfection complexes into endosomes/lysosomes which suggests an uptake of transfection complexes via endocytosis. It was also found that the uptake of transfection complexes by the cells was independent of the presence or absence of Ca2+ ions in the transfection medium. On the other hand, the presence of Ca2+ in the transfection medium dramatically changed the composition of the transfection complexes inside the endosome/lysosome compartment, which resulted in a strong reduction of H1 binding to DNA. Presence of Ca2+ in the postincubation medium for 24 h resulted in release of the transfection complexes with reduced H1 content from the endosomes/lysosomes into the cytosol. In the absence of Ca2+ the transfection complexes practically disappeared. These results allow us to come to the following conclusions: Ca2+ ions control the reorganization of the transfection complexes in endosomes/lysosomes and their release into the cytosol, which is an important prerequisite for transgene expression, whereas uptake of transfection complexes by the cells is not dependent on Ca2+.     

Microrheology and light transmission of blood

Employing both microscopic and photometric methods the rheology of pathological red cell aggregation was studied in model experiments. Suspensions of washed human red blood cells in dextran solutions containing rising concentrations of dextrans (M.W. 40000, 70000, 110000, 250000, 500000) were used. At low concentrations (less than 500 mg-%) of high molecular weight dextrans (greater than 70000) red cell suspensions formed aggregates similar to the ones found in normal human blood. At higher concentrations, the aggregates were similar to those observed in pathological human blood. The aggregates were studied under the condition of stasis, slow flow and at shear rate of their hydrodynamic dispersion. Besides, the flow behavior of the dispersed cells at high shear rates was studied. We found: 1. In all samples the rate of spontaneous aggregate re-formation in stasis (following hydrodynamic desaggregation) rose with rising dextran concentration up to 5.0 g-%. 2. The shear resistance of the aggregates, as measured by the shear stress necessary to keep them dispersed, rose up to concentrations of 2.5g-%, but fell at higher concentrations. 3. Only with dextran of a molecular weight above 110000 coarse agglomerates could be produced at high concentrations. Loose elastic meshes were rapidly produced at high concentrations of Dx 70. 4. When subjected to steady state low shear (m sec-1) only the agglomerates, but not the meshes rapidly grew in size. Most of the aggregation kinetics recorded by photometry and microscopy evaded detection by viscometry.     

Effects of conditioned stimulus pre-exposure on human electrodermal conditioning to fear-relevant and fear-irrelevant stimuli

The effects of conditioned stimulus (CS) pre-exposure and fear-relevance of the CS on human Pavlovian electrodermal conditioning were investigated. A differential delayed conditioning paradigm was used with a CS-unconditioned stimulus (US; shock) interval of 8 s. In Experiment 1, 64 subjects were randomized into four groups, two of which received fear-relevant stimuli and the other two fear-irrelevant stimuli. Half of the subjects were pre-exposed to the to-be-CSs and the other half to two not-to-be-CSs, with 15 exposure of each stimulus. During acquisition, subjects received 8 reinforced and 8 nonreinforced CS+ and CS- trials, and during the extinction phase 15 nonreinforced trials of each CS. Pre-exposure to the to-be-CSs retarded conditioning for the first and second interval anticipatory responses (FIRs and SIRs); that is, a latent inhibition effect was demonstrated, although the results for the FIR were inconclusive. The expected effects of fear-relevance were not revealed. Experiment 2 addressed the question whether the long pre-exposure period interfered with the frequently observed "preparedness effect" of higher resistance to extinction to fear-relevant stimuli. The design was similar to that of Experiment 1, but for half of the subjects the acquisition phase was initiated immediately after a short rest period, and for the other half acquisition started after an extended rest period, equal to the duration of the pre-exposure phase in Experiment 1. Twenty extinction trials of each CS were presented. A reliable difference in arousal in terms of spontaneous fluctuations was produced by the rest periods, but although differential conditioning was observed, no effect of fear-relevance was seen during extinction.     

Immunohistochemistry (S 100, KL 1) and human papillomavirus DNA hybridization on Morbus Bowen and bowenoid papulosis

Summary In this study 55 paraffin embedded samples defined as Bowen's disease or bowenoid papulosis were investigated with antibodies against S 100 protein and keratins (KL 1). S 100-positive cells were quantified and related to defined section area of the epidermal compartment by computer-assisted image analysis. The density of S 100-positive cells was compared with normal skin and was particularly related to growth patterns and keratinization of the different lesions under study. S 100-positive dendritic cells were found to be reduced overall in bowenoid lesions when compared with normal skin. Lesions with high counts of S 100-positive dendritic cells most frequentty showed a solitary growth pattern with highly conserved architecture and differentiation and no tendency to stromal invasion. In contrast, cases with low counts of S 100-positive cells very often showed multifocal development, a high degree of architectural disturbance and dedifferentiation. In this group, stromal invasion (cases of invasive carcinoma associated with Bowen's disease) was seen more often. Interestingly, this latter group of cases also revealed a peculiar keratin pattern. Frequently, the basal cell layer was decorated with KL 1 antibody, which usually recognizes only suprabasaly located keratinocytes. No differences between Bowen's disease and bowenoid papulosis were found in terms of densities of S 100-positive dendritic cells and keratin pattern. In our experience, extragenital Bowen's disease and genital Bowen's disease can not be distinguished on purely morphological grounds or with the immunocytochemical approach presented here. Interestingly, when employing in situ hybridization with HPV 16 probes three of seven samples of genital Bowen's disease harboured HPV 16 DNA, whereas six cases of extragenital disease were negative.Supported by the Deutsche Forschungsgemeinschaft (Lo 285/2-4)     

Regulation and possible physiological role of the Ca2-dependent K+ channel of cortical collecting ducts of the rat

In the luminal membrane of rat cortical collecting duct (CCD) a big Ca²⁺-dependent and a small Ca²⁺-independent K⁺ channel have been described. Whereas the latter most likely is responsible for the K⁺ secretion in this nephron segment, the function of the large-conductance K⁺ channel is unknown. The regulation of this channel and its possible physiological role were examined with the conventional cell-free and the cell-attached nystatin patch-clamp techniques. Patch-clamp recordings were obtained from the luminal membrane of isolated perfused CCD segments and from freshly isolated CCD cells. Intracellular calcium was measured using the calcium-sensitive dye fura-2. The large-conductance K⁺ channel was strongly voltage- and calcium-dependent. At 3 mol/l cytosolic Ca²⁺ activity it was half-maximally activated. At 1 mmol/l it was neither regulated by cytosolic pH nor by ATP. At 1 mol/l Ca²⁺ activity the open probability (P _o) of this channel was pH-dependent. At pH 7.0 P _o was decreased to 4±2% (n=9) and at pH 8.5 it was increased to 425±52% (n=9) of the control. At this low Ca²⁺ activity the P _o of the channel was reduced by 1 mmol/l ATP to 8±4% (n=6). Cell swelling activated the large-conductance K⁺ channel (n=14) and hyperpolarized the membrane potential of the cells by 9±1 mV (n=23). Intracellular Ca²⁺ activity increased after hypotonic stress. This increase depended on the extracellular Ca²⁺ activity. A possible physiological function of the large-conductance K⁺ channel in rat CCD cells may be the reduction of the intracellular K⁺ concentration after cell swelling. Once this channel is activated by increases in the cytosolic Ca²⁺ activity it can be regulated by changes in cellular pH and ATP.Supported by DFG Schl 277/2-3     

Surface markers of human natural killer cells as analyzed in a modified single cell cytotoxicity assay on poly-L-lysine coated cover slips

A modified single cell cytotoxicity assay using poly-L-lysine coated cover slips (PLL-SCCA) was employed to study the frequency and surface marker profile of human peripheral blood lymphocytes (PBL) with NK reactivity against K 562 target cells. When compared with the previously described agarose single cell cytotoxicity assay (A-SCCA) identical results were obtained. For 13 donors tested 18.1 +/- 4.4% of the PBL formed conjugates with K 562 and 2.7 +/- 1.6% displayed NK reactivity. In contrast to the A-SCCA, the PLL-modified assay permits direct identification of both conjugate forming (TBC) and cytolytic PBL (NK) by means of surface markers. Indirect immunofluorescence studies with monoclonal anti-PBL antibodies revealed that neither the plating procedures nor the incubation conditions employed affected the expression of the antigens recognized by these reagents. This method of directly identifying NK cells showed that OKM1+ cells were enriched among the NK cells as compared to PBL and TBC (55% vs. 23% and 43%, respectively). In contrast, the OKT3+ or Leu1+ fraction of the NK cells was reduced as compared to PBL and TBC. However, using this method of identification at the effector cell level, a substantial proportion of the NK cells were OKT3+ or Leu1+ (57% or 58% respectively, 7 donors). Approximately 25% of the NK cells were Leu2a+ and 30% were Leu3a+, respectively. However, the size of the Leu3a+ fraction varied considerably with individual donors and the size of this fraction appeared to be inversely related to that of the donors NK pool.     

Lack of correlation between impaired Interferon production and natural killer activity of lymphocytes in multiple sclerosis

Summary The ability of lymphocytes from patients with multiple sclerosis (MS) to produce Interferon (IFN-) in response to Newcastle Disease Virus (NDV) was studiedin vitro. The correlation between individual IFN- titers and natural killer (NK) cell activity and the presence of HLA system antigens associated with MS (B-7 and DRW-2) was also investigated.Lymphocytes from MS patients showed a significantly impaired capacity to synthesize IFN-in vitro when compared to lymphocytes from healthy donors (mean titers: 85.9 I.U. and 268.2 I.U., respectively). Marked differences in IFN- titers were observed in the group of MS patients.The production of IFN- by the patients' lymphocytes did not correlate with either the activity of NK cells or with their stimulation by exogenous IFN-. There was also no correlation between IFN- production by lymphocytes from MS patients and the presence or absence of B-7 and DRW-2 antigens.With 3 Figures     

Pancreatic glucagonoma with and without syndrome

Summary In five patients single or multiple glucagonomas were characterized by immunocytochemistry. Two large single glucagonomas were associated with the glucagonoma syndrome, which completely dissappeared after removal of the tumours. The morphologic findings in these patients are compared with 48 others collected from literature.In the other three patients, the glucagonomas were not associated with a clinical syndrome and were detected by chance (one accompanying an insulinoma; the other in pancreases of patients suffering from multiple endocrine neoplasia I; MEN I). These tumours appeared by their histological, immunocytochemical and ultrastructural features better organized than the glucagonomas with syndrome.Glucagonomas not producing a syndrome can be classified into (a) solitary, often malignant endocrine pancreatic tumours, (b) glucagonomas associated with insulinomas and other tumours, (c) multiple glucagonomas in MEN I and (d) single microglucagonomas in elderly patients. It is emphasized that only immunohistology allows clear identification of these tumours as glucagonomas.