k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.     

Heterogeneity of hepatic microsomal UDP-glucuronosyltransferase(s) activities: comparison between human and mammalian species activities

The first comparative profiles of UDP-glucuronosyltransferase(s) (UDPGT) activities obtained under standard conditions in vitro in mammals (man, rat [Wistar and Gunn], mouse, monkey [Papio papio and Cynomolgus], pig, guinea pig, rabbit, dog) are presented for 16 aglycones. A decreasing scale of these activities was obtained from planar to bulky molecules. The scale was identical for each of the mammals studied, including man. Statistical analysis of the results revealed a division of the aglycones into three groups, one being correlated with the molecular form called GT1 the two others with the GT2 form. The profile of activities in the Gunn rat revealed very weak activity towards planar molecules (GT1). These results provide evidence that under standard conditions, human UDPGT activities are comparable to those from other animals.     

Dye coupling in the organ of Corti

Summary Dye-coupling in an in vitro preparation of the supporting cells of the guinea-pig organ of Corti was evaluated by use of the fluorescent dyes, Lucifer Yellow, fluorescein and 6 carboxyfluorescein. Despite the presence of good electrical coupling in Hensen cells (coupling ratios >0.6) the spread of Lucifer yellow was inconsistent. Hensen cells are very susceptible to photoinactivation, i.e., cell injury upon illumination of intracellular dye; and this in conjunction with Lucifer Yellow's charge and K+-induced precipitability may account for its variability of spread. Fluorescein and 6 carboxyfluorescein, on the other hand, spread more readily and to a greater extent than Lucifer Yellow, often spreading to cell types other than those of Hensen. Dye spread is rapid, occurring within a few minutes. These results suggest that molecules of metabolic importance also may be shared by the supporting cells of the organ of Corti.     

Correlation of neuronal size and peptide immunoreactivity in the guinea-pig trigeminal ganglion

Summary Frequency and size of guinea-pig trigeminal neurones immunoreactive with antisera to -neo-endorphin(-neo-END), dynorphin A-(DYN), vasoactive intestinal polypeptide-(VIP), somatostatin-(SOM), and substance P-(SP) are reported. Co-localisation of the various peptides to the same ganglion cells was investigated immunocytochemically in consecutive 7-m thick paraffin sections. According to their size, all peptide-immunoreactive neurones belong to the class of small ganglion cells. Within this cell group, SP-immunoreactive neurones appear to be the largest, followed by SOM-, VIP-, -neo-END- and DYN-immunoreactive ganglion cells. The observed differences in size are statistically significant with the exception of that between -neo-END and DYN. This finding correlates well with the observed co-occurrence of the two immunoreactive peptides. All -neo-END-immunoreactive perikarya are also reactive to VIP antisera. These neurones are significantly smaller than those containing VIP-immunoreactivity exclusively. Ganglion cells displaying co-existence of -neoEND- and SP-immunoreactivity or VIP- and SP-immunoreactivity are found too infrequently to allow morphometric analysis. Some non-immunoreactive ganglion cells are shown to be approached by dense baskets of VIP-, -neo-END- or SP-immunoreactive varicose fibres, indicating the presence of intraganglionic modulation sites. The combination of immunohistochemistry and morphometry presented in this study allows the differentiation of diverse populations of primary afferent neurones exhibiting peptide immunoreactivity, most likely reflecting their involvement in different central and peripheral reflex arcs and sensory modalities.     

Localization of substance P-like immunoreactive fibers in the thoracic spinal cord of guinea pig

Summary A dorsal-horn fiber system is revealed in the thoracic spinal cord of guinea pig by means of substance P immunocytochemistry. This system has repeated craniocaudal and/or caudo-cranial extensions and possesses five main components: (1) a superficial network, situated beneath the dorsolateral surface of the spinal cord. This network is connected with the dorsal root fibers and the accumulations of substance P-like immunoreactive (SP-LI) fibers in the Lissauer's tract; (2) an accumulation of SP-LI fibers in the Lissauer's tract at the border of the dorsal horn; (3) two collateral SP-LI fascicles (one lateral and one medial) emerging from the SP-LI fiber accumulation in the Lissauer's tract; (4) a transversal fascicle running through laminae III–V, and (5) an SP-LI network in the region of the lateral spinal cord nucleus. These components of the dorsal-horn fiber system show widespread connections with ipsi-and contralateral spinal cord areas, connecting them in cranio-caudal and/or caudo-cranial directions. The SP-LI dorsal-horn system has close relationship with groups of preganglionic sympathetic cells in the intermediate zone of the spinal cord, respective with the vegetative network of this zone. It is suggested that some fibers of the dorsal-horn system that originate from dorsal-root ganglia may represent primary sensory or visceral afferents. It is likely that the dorsal-horn fiber system and the vegetative network of the thoracic spinal cord may represent the morphological basis for the integration of (1) the central and peripheral vegetative nervous systems, and (2) the somatic and vegetative nervous system.     

Size fractionation of thermal aggregates of immunoglobulin G

Purified pooled human immunoglobulin G (IgG) in solution, when extensively heated at high temperatures or for long periods, irreversibly aggregates and insoluble precipitates result. However, when IgG solutions are heated in the temperature range 55-65 degrees C for more limited time periods, soluble turbid polydispersed aggregate mixtures are obtained. Gel filtration of such aggregate mixtures on calibrated Bio-Rad A-150m columns demonstrates a continuous size distribution from dimers to aggregates as large as 4 X 10(7) Da (200-mers) with no particular size predominant. Chromatographically reproducible cuts of narrow size heterogeneity can be obtained by short-time fraction collection. Elution-time reproducibility is excellent both for mixture and for individual cuts. Stability studies indicate that reproducible and stable aggregates may be made from purified IgG and that fractionated aggregates should be stored quick-frozen until needed. Sized IgG aggregates have proved useful in reactivity studies with rheumatoid factor, animal anti-IgG antibodies, and complement.     

Modification of the zona-free hamster ova bioassay of boar sperm fertility and correlation with in vivo fertility

These studies were designed to evaluate the ability of the zona-free hamster ova bioassay to detect differences in fertility of boar sperm. In the first study, sperm from two previously infertile boars were compared to sperm from seven previously fertile boars. The percentage of zona-free hamster ova penetrated by sperm from the previously infertile boars was significantly lower than the percentage of ova penetrated by sperm from previously fertile boars (18% of ova penetrated vs. 83%, P < .001). In the 14 ejaculates from the previously infertile boars that had ejaculate motilities of 50% or greater, the percentage of zona-free hamster ova penetrated continued to be lower than in ejaculates from the fertile boars. One of the two previously infertile boars consistently had a normal semen analysis. The only two observed manifestations of his reduced fertility were his zero conception rate and the limited ability of his sperm to penetrate zona-free hamster ova. In the second study, females were inseminated with equal numbers of sperm from two previously fertile males and the paternity of offspring determined at birth. The experiment was replicated with four combinations of six boars. A high correlation was observed between the percentage of offspring sired and the ability to penetrate zona-free hamster ova (R = .89). Neither morphology nor the ability of the sperm to undergo an acrosome reaction during in vitro incubation was correlated with fertility in the competitive mating situation. These results suggest the zona-free hamster ova bioassay can improve the in vitro fertility assessment of fresh boar semen.     

中国家猪高分辨G—带及模式图

采用氨甲喋呤或胸苷阻断法使细胞分裂同步化,并结合胰酶G-带技术,对中国7个家猪品种高分辨G-带进行了研究,发现家猪品种间带型基本一致,从而参照人类细胞遗传学命名法的国际体制,提出了中国家猪高分辨G-带标准化核型及模式图,对显带核型界标进行了少许修改,对每对染色体进行了区带划分和描述。单倍染色体组所显示的G-带数目,包括X和Y染色体,巳达444条,近于中期染色体带纹数目的两倍。     

The relationship between serum and cell type on the development of rat and pig cultured preadipocytes

Stromal-vascular cells from rats and pigs were isolated from adipose tissue and used to measure preadipocyte proliferation and differentiation. Cells from rats and pigs were grown in either 2.5% pig serum or 2.5% rat serum. Cells were either supplemented or unsupplemented with insulin after five days of growth in culture. In these cultures, pig fat cells developed as discrete clusters while rat fat cells developed as loose clusters or as individual cells. Rat cells had greater levels of sn-glycerol phosphate dehydrogenase activity compared to pig cells. Rat serum increased soluble protein in plated cells when compared to cells grown in pig serum. Pig serum increased glycerol phosphate dehydrogenase specific activity when compared to rat serum. In this system, there was no response to insulin. The cells grown in rat serum did not resemble adipocytes in regard to the presence of large lipid droplets (oil red 0 staining). These results demonstrate that rat and pig stromal-vascular cells in culture are morphologically different. Cells from both species, however, responded similarly to sera from either species showing that cells from rats and pigs responded to the growth and differentiation factors present in these sera.Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Cell-free transfer of sterols from dictyosome-like structures to plasma membrane vesicles of guinea pig testes

Summary Transfer of radiolabeled lipids from dictyosome-like structures (DLS) from testis tubules of the guinea pig as donor to unlabeled plasma membrane from testis tubules immobilized on nitrocellulose as acceptor was studied in a completely cell-free system. As a general label for lipids of the donor DLS, isolated testis tubules were incubated with [¹⁴C]acetate. Time- and temperature-dependent transfer of [¹⁴C]acetate labeled constituents was observed in the cellfree system. However, despite the fact that phospholipids and other constituents were highly labeled in the donor fraction, primarily radioactive sterols were transferred to the plasma membrane acceptor vesicles. Transfer at 37°C represented 0.4 to 0.7% of the total radiolabeled cholesterol at 37°C but little or no transfer occurred at 4°C. The sterols transferred exhibited Chromatographic mobilities corresponding to those of cholesterol and lanosterol. Similar results were obtained with [¹⁴C]mevalonic acid. In subsequent experiments, cholesterol transfer from DLS to plasma membrane was demonstrated by incubation of DLS with [³H]squalene which was converted into sterol or with [¹⁴C]cholesterol. Transfer of sterols required ATP, but not cytosol, and was both time- and temperature-dependent. DLS were more effective than either endoplasmic reticulum or plasma membrane as the donor fraction. The results from the cell-free analysis suggest a possible functional role of the DLS in sterol biogenesis and transfer to the plasma membrane during spermatid development.Abbreviations DLS dictyosome-like structure(s) - PBS phosphatebuffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin