Histochemical localization of carbonic anhydrase in the testis and epididymis of the rabbit.

The distribution of carbonic anhydrase (CA) was studied in the testis and epididymis of mature, male rabbits using a cobalt precipitation method. CA was found only in the endothelium of the capillaries in the testis. The epididymal duct was divided into initial, middle and terminal segments. Strong cytoplasmic CA was present in the apical cells in the initial and middle segments. Vacuoles with CA staining in the membranes were found in the principal cells in the middle segment. Intensely stained basal cells were present in the terminal segment. In the last part of the terminal segment and the first of the ductus deferens the basolateral cell membranes were also stained. The function of the enzyme is discussed especially in relation to acidification of the epididymal fluid and facilitation of CO2 diffusion.     

Methods for determining the proliferation kinetics of cells by means of 5-bromodeoxyuridine.

After treatment of cells with 5-bromodeoxyuridine (BUdR), the percentage of completely BUdR-labelled interphase nuclei is greater the longer the BUdR treatment. The labelling effect is visible after staining with the fluorochrome 33258 Hoechst and with Giemsa. Various formulae and a nomogram are presented by means of which the percentage of cells in S period, duration of the S period and the whole cell cycle can be determined by examination of a single preparation or by comparison of several preparations. The methods are tested using cell cultures of Microtus agrestis and compared with autoradiographic methods after labeling with 3H-thymidine.     

Localization of carbonic anhydrase activity in the developing boar testis

Embryonic and fetal pig gonads were obtained immediately after the sow's slaughter at 18, 21, 25, 28, 30, 36, 55, 63, 80 or 108 days of pregnancy. Semithin plastic sections were incubated for localization of carbonic anhydrase (CA) activity using a cobalt precipitation technique. In the embryonic gonad, CA activity was only present in the coelomic epithelium and in the endothelium of scattered blood capillaries. In the early testes (30-36 days) the CA activity was also localized in the cytoplasm of the sustentacular cells. Both spermatogonia and the developing interstitial cells were negative. At later stages, the testes presented a clear CA cytoplasmic activity in the Sertoli cells and a membrane-bound activity in the peritubular capillaries, resembling the enzymatic localization in the adult. The epithelium of the rete testis had a clear membrane-bound CA activity. CA histochemistry is useful as a marker for topographical studies of Sertoli cells during the prenatal development in the pig.     

Suppression of a thymus dependent humoral response in mice by concanavalin A in vivo

Mice treated with Concanavalin A prior to immunization with sheep erthyrocytes exhibit a markedly reduced plaque forming spleen cell response. This immunosuppressive effect could be reversed by using higher doses of antigen or priming the animals with nonimmunizing doses of antigen prior to Concanavalin A injection designed to either by-pass or enhance thymus derived lymphocyte functions. It was also demonstrated that Concanavalin A in vivo activated the thymus derived lymphocyte subpopulation in the spleen, and this activation was dose dependent and correlated with the immunosuppression observed. Animals injected with Concanavalin A at various times prior to whole body lethal irradiation would not support the plaque forming cell response of adoptively transferred normal syngeneic spleen cells. This effect was shown to be time and dose of Concanavalin A dependent. It was also shown that the route of injection of Concanavalin A prior to irradiation determined the results observed, in that the intravenous route resulted in the suppression of transferred cells, while the intraperitoneal route showed no effect. It is suggested that Concanavalin A induced immunosuppression of the humoral, thymus dependent immune response in mice results for the activation of a subpopulation of thymus derived suppressors cells, and that the effect is short lived, radiation resistant, and dose of Concanavalin A and antigen dependent.     

The effectiveness of flower strips and hedgerows on pest control,pollination services and crop yield: a quantitative synthesis

Floral plantings are promoted to foster ecological intensification of agriculture through provisioning of ecosystem services. However, a comprehensive assessment of the effectiveness of different floral plantings, their characteristics and consequences for crop yield is lacking. Here we quantified the impacts of flower strips and hedgerows on pest control (18 studies) and pollination services (17 studies) in adjacent crops in North America, Europe and New Zealand. Flower strips, but not hedgerows, enhanced pest control services in adjacent fields by 16% on average. However, effects on crop pollination and yield were more variable. Our synthesis identifies several important drivers of variability in effectiveness of plantings: pollination services declined exponentially with distance from plantings, and perennial and older flower strips with higher flowering plant diversity enhanced pollination more effectively. These findings provide promising pathways to optimise floral plantings to more effectively contribute to ecosystem service delivery and ecological intensification of agriculture in the future.     

Index hopping on the Illumina HiseqX platform and its consequences for ancient DNA studies

The high‐throughput capacities of the Illumina sequencing platforms and the possibility to label samples individually have encouraged wide use of sample multiplexing. However, this practice results in read misassignment (usually <1%) across samples sequenced on the same lane. Alarmingly high rates of read misassignment of up to 10% were reported for lllumina sequencing machines with exclusion amplification chemistry. This may make use of these platforms prohibitive, particularly in studies that rely on low‐quantity and low‐quality samples, such as historical and archaeological specimens. Here, we use barcodes, short sequences that are ligated to both ends of the DNA insert, to directly quantify the rate of index hopping in 100‐year old museum‐preserved gorilla (Gorilla beringei) samples. Correcting for multiple sources of noise, we identify on average 0.470% of reads containing a hopped index. We show that sample‐specific quantity of misassigned reads depends on the number of reads that any given sample contributes to the total sequencing pool, so that samples with few sequenced reads receive the greatest proportion of misassigned reads. This particularly affects ancient DNA samples, as these frequently differ in their DNA quantity and endogenous content. Through simulations we show that even low rates of index hopping, as reported here, can lead to biases in ancient DNA studies when multiplexing samples with vastly different quantities of endogenous material.     

In Vitro and In Vivo Evaluation of a 18F-Labeled High Affinity NOTA Conjugated Bombesin Antagonist as a PET Ligand for GRPR-Targeted Tumor Imaging

Expression of the gastrin-releasing peptide receptor (GRPR) in prostate cancer suggests that this receptor can be used as a potential molecular target to visualize and treat these tumors. We have previously investigated an antagonist analog of bombesin (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂, RM26) conjugated to 1,4,7-triazacyclononane-N,N'',N''''-triacetic acid (NOTA) via a diethylene glycol (PEG₂) spacer (NOTA-P2-RM26) labeled with ⁶⁸Ga and ¹¹¹In. We found that this conjugate has favorable properties for in vivo imaging of GRPR-expression. The focus of this study was to develop a ¹⁸F-labelled PET agent to visualize GRPR. NOTA-P2-RM26 was labeled with ¹⁸F using aluminum-fluoride chelation. Stability, in vitro binding specificity and cellular processing tests were performed. The inhibition efficiency (IC₅₀) of the [^natF]AlF-NOTA-P2-RM26 was compared to that of the ^natGa-loaded peptide using ¹²⁵I-Tyr⁴-BBN as the displacement radioligand. The pharmacokinetics and in vivo binding specificity of the compound were studied. NOTA-P2-RM26 was labeled with ¹⁸F within 1 h (60-65% decay corrected radiochemical yield, 55 GBq/µmol). The radiopeptide was stable in murine serum and showed high specific binding to PC-3 cells. [^natF]AlF-NOTA-P2-RM26 showed a low nanomolar inhibition efficiency (IC₅₀=4.4±0.8 nM). The internalization rate of the tracer was low. Less than 14% of the cell-bound radioactivity was internalized after 4 h. The biodistribution of [¹⁸F]AlF-NOTA-P2-RM26 demonstrated rapid blood clearance, low liver uptake and low kidney retention. The tumor uptake at 3 h p.i. was 5.5±0.7 %ID/g, and the tumor-to-blood, -muscle and -bone ratios were 87±42, 159±47, 38±16, respectively. The uptake in tumors, pancreas and other GRPR-expressing organs was significantly reduced when excess amount of non-labeled peptide was co-injected. The low uptake in bone suggests a high in vivo stability of the Al-F bond. High contrast PET image was obtained 3 h p.i. The initial biological results suggest that [¹⁸F]AlF-NOTA-P2-RM26 is a promising candidate for PET imaging of GRPR in vivo.