Vorwort

Ohne Zusammenfassung     

Ernest H. Starling

Ohne Zusammenfassung     

Chromatographic techniques for the separation of Al and associated organic ligands present in soil solution

Organic acids including humic, fulvic, aliphatic and aromatic acids comprise part of the dissolved organic carbon (DOC) present in soil solution. They act as ligands for trace metals and are effective detoxifiers of monomeric aluminium (Al). Solid phase extraction (SPE) techniques permit fractionation of the DOC into organic classes but yield no information on the pre-existing Al/organic acid complexes. Aliphatic and aromatic acids may be separated and determined by High Performance Liquid Chromatography (HPLC); however, the conditions used dissociate the organic acid Al complexes. Humic and fulvic acids are of a variable and ill-defined nature and only limited information exists regarding their binding of Al. This paper reports on fractionation studies of soil solutions, using both SPE and molecular weight cut-off filters, to characterise the DOC components and on the subsequent development of a size exclusion chromatography (SEC) system for the separation of organically complexed Al into different species using a low ionic strength mobile phase at pH 4.2. Model complexes of Al and Cu citrate were used to evaluate chromatographic performance of a Fractogel TSK HW-40(S) column (1×30 cm). For soil solution samples, the column eluate, after passing through a UV detector, was directly coupled to an Inductively Coupled Plasma Atomic Emission Spectrometer (ICPAES) for on-line multi-element detection to characterise DOC and trace metal distribution.Fractionation studies revealed that polysaccharides constituted the major proportion of the DOC which passed the 10000 dalton molecular weight cut-off filter. Analysis of soil solutions from an organically amended soil by the SEC-ICPAES system showed that Al, Fe and Mn eluted as multiple peaks prior to the bed volume, indicating their presence as complexes with organic ligands.     

Successful in vitro culture of early cleavage stage embryos recovered from superovulated red deer (Cervus elaphus)

Three separate embryo culture systems were evaluated for their ability to support development of early cleavage stage red deer (Cervus elaphus ) embryos: ligated sheep oviducts (Treatment A); cervine oviduct epithelial monolayer in TCM 199 + 10% deer serum (Treatment B); synthetic oviduct fluid + 20% human serum at 7% O(2) atmosphere (Treatment Q. In addition, 2 superovulation protocols were compared for their efficacy in producing early cleavage stage embryos. Twenty red deer (2 to 7 yr old) were synchronized in April with intravaginal CIDR devices for 12 d. All animals received a total of 0.4 units of ovine FSH administered in 8 equal doses, 12 h apart, beginning 72 h before removal of CIDR devices. The deer additionally received 200 IU PMSG, either with the first FSH injection (Group 1, n = 10) or with the last FSH injection (Group 2, n = 10). Hinds were placed with fertile stags following withdrawal of CIDR devices. Ova were collected by surgical recovery 63 h post CIDR removal. At the time of collection, animals in Group 2 had a significantly greater mean (+/- SEM) ovulation rate (11.2 +/- 2.4 vs 5.3 +/- 2.4), with more animals responding to treatment (>1 ovulation), than the animals in Group 1 (10/10 vs 4/10). Late in the breeding season (June), 10 additional red deer (Group 3, Experiment 2) were superovulated using the same protocol as for the deer in Group 2, with ova collection advanced by 24 h. Mean (+/- SEM) ovulation rate was 6.4 +/- 1.2 with 9 10 animals responding. Ova recovery did not differ among the groups (range 73 to 87%). Superovulation treatment did not affect cultured embryo development to the morula/blastocyst stage. Furthermore, there was no difference among the 3 culture systems in their support of development either to the morula (range 50 to 58%) or to the blastocyst (range 22 to 26%) stage. After laparoscopic transfer of 4 morula/blastocyst embryos to recipient red deer (2 from Treatment B and 2 from Treatment C) 2 live calves were born from embryos cultured in Treatment B.     

Ants as bioindicators of habitat disturbance: validation of the functional group model for Australia's humid tropics

A functional group model of ant community composition has been widely used in Australia to analyse biogeographical patterns of ant community structure and the responses of ant communities to disturbance. The model has provided valuable support to the widespread use of ant communities as bioindicators of ecological change. However, the model was developed from studies of arid-zone faunas, and its applicability to the World Heritage rainforests of Queensland's humid tropics has not yet been validated. Here we test predictions based on the functional group model for ant communities in Queensland's humid rainforests, by documenting ant community composition and its responses to disturbance on the Atherton Tablelands. Five sites were studied, comprising two relatively undisturbed reference sites representing contrasting rainforest types, and three previously cleared sites, two of which were undergoing revegetation. A variety of sampling techniques were employed, including pitfall trapping, litter extractions, baiting, and general searching. A total of 50 ant species from 29 genera were collected. Site species richness was highest at the reference sites, and lowest at the unvegetated disturbed site, and overall was negatively related to mean ground temperature. As predicted by the functional group model, behaviorally dominant dolichoderines were uncommon or absent at the reference sites, and the most common ants were Generalized myrmicines and Opportunists. Also as predicted, habitat disturbance favored Opportunists, and, as the disturbance involved canopy clearance, this led to colonization by Iridomyrmex and other Dominant dolichoderines. Opportunists represented about 40% of total ants in traps at the reference sites, compared with 80–95% at the disturbed sites. Except one species, Tropical Climate Specialists and Specialist Predators were absent from disturbed sites.In conclusion, patterns of ant composition in relation to disturbance on the Atherton Tablelands conform to the functional group model that has been widely applied to ant faunas elsewhere in Australia. The model may therefore play an important role in the use of ants as bioindicators of ecological change in the World Heritage rainforests of this region.     

Role of catabolite regulatory mechanisms in control of carbohydrate utilization by the rumen anaerobic fungus Neocallimastix frontalis.

Neocallimastix frontalis PN-1 utilized the soluble sugars D-glucose, D-cellobiose, D-fructose, maltose, sucrose, and D-xylose for growth. L-Arabinose, D-galactose, D-mannose, and D-xylitol did not support growth of the fungus. Paired substrate test systems were used to determine whether any two sugars were utilized simultaneously or sequentially. Of the paired monosaccharides tested, glucose was found to be preferentially utilized compared with fructose and xylose. The disaccharides cellobiose and sucrose were preferentially utilized compared with fructose and glucose, respectively, an cellobiose was also the preferred substrate compared with xylose. Xylose was the preferred substrate compared with maltose. In further incubations, the fungus was grown on the substrate utilized last in the two-substrate tests. After moderate growth was attained, the preferred substrate was added to the culture medium. Inhibition of nonpreferred substrate utilization by the addition of the preferred substrate was taken as evidence of catabolite regulation. For the various combinations of substrates tested, fructose and xylose utilization was found to be inhibited in the presence of glucose, indicating that catabolite regulation was involved. No clear-cut inhibition was observed with any of the other substrate combinations tested. The significance of these findings in relation to rumen microbial interactions and competitions is discussed.     

Effects of pinealectomy on reproduction in the Syrian hamster mutant anophthalmic white (Wh)

The gene Wh, causing anophthalmia in the Syrian hamster, Mesocricetus auratus, is a pleiotropic gene affecting eye development, pigmentation, hearing, and reproduction. Male hamsters homozygous for this gene are usually sterile. Since both Wh and the pineal organ are known to suppress reproductive function, the objective of this study was twofold: (1) to determine whether Wh, by itself, influences testicular differentiation; and (2) to determine whether removal of the pineal gland will restore fertility to both experimentally blinded (B), genetically normal [wh/wh(B)] hamsters and mutant, eyeless (Wh/Wh) hamsters. Accordingly, one testis from each of ten wh/wh(B) and ten Wh/Wh hamsters at approximately 60 days of age was removed, and these testes were compared at the gross and light microscopic level. Since all testes were identical at 60 days of age and contained normal differentiating germ cells, the gene Wh does not appear to affect initial testicular differentiation. Testicular tissues from at least ten wh/wh, wh/wh(B), heterozygous (Wh/wh), and Wh/Wh hamsters, at 135 days of age, were also compared. Testes from all wh/wh(B), and 70% of the Wh/Wh hamsters were hypoplasic and aspermic. Approximately 30% of the testes from Wh/Wh hamsters contained some seminiferous tubules with normal sperm present. Pinealectomy fully restored adult testicular size and morphology in all wh/wh(B) and Wh/Wh hamsters. Thus, it was demonstrated that the atrophy of testes from Wh/Wh individuals is a pineal-mediated phenomenon due to failure of eye development and the subsequent lack of a functional visual pathway. Testes from Wh/Wh hamsters appear to be completely competent to respond to the normal, antigonadotrophic effects of the pineal.     

Ultraviolet resonance Raman and absorption difference spectroscopy of myoglobins: titration behavior of individual tyrosine residues

The UV resonance Raman spectra of horse and sperm whale myoglobin excited at 240 nm show bands between 600 and 1700 cm-1 which derive from tyrosyl and tryptophyl residues. No significant contribution from phenylalanine and peptide backbone vibrations occurs at this excitation wavelength. We examine the pH dependence of the UV resonance Raman and UV absorption difference spectra of these myoglobins to correlate the local protein environment of the tyrosyl residues as given by the protein crystal structure to their pKa values, molar absorptivities, and Raman cross sections. Some of our pKa values for the tyrosinate residues of horse Mb differ from those of previous studies. We show that the lambda max values, the molar absorptivities, and the Raman cross sections are sensitive to the local environment of the tyrosinate residues in the protein. We relate differences in the tyrosyl absorption spectra to differences in Raman cross sections. In addition, we discuss the importance to the Raman cross sections of the local electromagnetic field enhancement due to the dielectric environment of the tyrosinate residues in the protein. This local field should scale the Raman cross sections in a way useful as a probe of the average aromatic amino acid residue environment.     

Defective presentation of endogenous antigens by a murine sarcoma. Implications for the failure of an anti-tumor immune response

MHC class I-restricted CTL play a central role in the immune response against methylcholanthrene (MCA)-induced sarcomas in mice. We, therefore, hypothesized that MCA-induced tumors may evade immune recognition by failing to present Ag to CD8+ CTL. Of a number of previously described MCA-induced sarcomas, one, MCA 101, fails to induce CTL, is nonimmunogenic, and grows rapidly and lethally in nonimmunosuppressed recipients. To better understand the nonimmunogenicity of MCA 101 we examined its ability to present foreign Ag to CTL. Unlike immunogenic sarcomas, MCA 101 failed to present endogenously synthesized influenza virus Ag to influenza virus-specific CTL. The deficiency in presentation of endogenous Ag by MCA 101 was attributed to a markedly reduced rate of synthesis of class I molecules because up-regulation of class I synthesis by IFN-gamma greatly increased the presentation of influenza A virus Ag. Despite low levels of cell surface class I expression, MCA 101 presented exogenous peptide Ag to anti-influenza CTL with efficiency similar to immunogenic MCA sarcoma cell lines. These findings could not be attributed to deficiencies in class I assembly or transport, as has been suggested by others who have studied mutant cells with defective Ag presentation. Furthermore, our studies suggest that some tumor cells can escape recognition by CTL and subsequent immune eradication by suppressing presentation of endogenous Ag.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.