Histoenzymatic characterization of sub-types of Type I fibres in fast muscles of rats

Summary The histochemical activities of succinic dehydrogenase (SDH), myofibrillar Adenosine triphosphatase (ATPase) and alpha glycerophosphate dehydrogenase were studied in serial sections of rat vastus lateralis (red) (RVL), gastrocnemius and diaphragm muscles. Three main fibre-types were distinguished. The Type I fibres of RVL and gastrocnemius muscles fell into two distinct groups: one category-Type IA showed very low ATPase activity. The second category of Type IB fibres displayed moderate ATPase reaction. The Type IA fibres were divisible into two sub-groups when tested for SDH reaction. Type IA₁ fibres possessed a homogenous distribution of diformazan·granules throughout the fibre: Type IA₂ fibres displayed characteristic moth-eaten pattern of diformazan localization. The diaphragm muscle did not show either Type IB or Type IA₂ varieties. The great majority of Type I fibres were sub-type IA₁ in the three fast muscles studied. It is also demonstrated here that an inherent heterogeneity exists between Type I fibres of diaphragm and leg muscles in regard to -GPD localization. This histochemical data emphasizes the fact that subdivision of Type I striated muscle fibres of mammalian animals into two sub-types is only approximate and that a further subcategorization is possible.     

A quantitative radioimmunoassay for membranous and soluble H-Y antigen typing

Summary Two sensitive and quantitative methods for membranous or soluble H-Y antigen typing using rat anti-H-Y immune sera and ¹²⁵I labelled protein A were carried out.These techniques were used to study H-Y antigen expression in human cell lines, and to refine the hypothesis that ( ₂m) serves as an anchorage point for H-Y antigen.     

The definition of a new IA antigenic specificity using the B6.C-H-2 bm12 I-region mutant strain

A new Ia specificity has been defined using theIA-subregion mutant B6.C-H-2 ^bm12. The immunization to produce the antiserum wasbm12 anti-A.BY, as all other immunizations, such asbm12 anti-C57BL/6, failed to produce antibody. By selecting strains of C57BL origin for testing, it was shown that, (a) the serum was only weakly cytotoxic but gave substantial reactions using a rosetting assay; (b) the antibody reacted with B cells and not T cells; (c) strains of theb, d, p andq H–2 haplotypes were positive, whereasf, k, r ands were negative; (d) absorption studies demonstrated only a single specificity to be present and by testing recombinant strains, the reaction mapped to theIA subregion; (e) SDS-PAGE demonstrated that the antiserum reacted with a molecule of MW 33 000. Preliminary studies indicate this new specificity, present on C57BL/6 and lost frombm12, is present on the same molecule as other I-A specificities.This work was supported by funds obtained from the N. H. and M. R. C. (Australia) and the National Institutes of Health, Grant No. CA-21224.     

The mechanism of object fixation and its relation to spontaneous pattern preferences in Drosophila melanogaster

The orientation behavior of walking flies, Drosophila melanogaster, towards a single 6° wide black vertical stripe (elementary stripe) can be explained by use of the turning tendency function H(). This function is characterized by maximal values at an angular distance of =25° from the stable zero position (=orienting direction), a sharp decline from this maximum to =60°, and a very slow approach to the unstable zero position (Horn and Wehner, 1975). The shape of this function is influenced by both translatory and rotatory components of movement. If the translatory component is minimized by measuring the turning function W() (see 2.3) at a distance of 10 mm (C1) from the center of the arena, a change in the strength of this decline is caused. But with increasing translatory component, i.e. at a greater distance from the center of the arena, W() approximates the heuristical function H() (Fig. 12). The turning functions W() are pattern-specific; the angular positions of the maximum responses shift to greater angles with increasing width of the patterns (Fig. 2). In the twopattern configuration with double or single stripes, there is always a coincidence between the stable zero positions of W (), the mean of the frequency distributions P() of the flies' positions and n _g() of the straight courses, and the stable zero positions of H () obtained from an additive superposition of two or more angular shifted turning tendency functions H() (Fig. 5, 7). Therefore, the mean positions of the flies in a multi-stripe experiment composed of elementary stripes can be predicted from the addition of many angular shifted turning tendency functions H(). Between H() and the frequency distribution P() of the flies' positions , the following formula holds: P() =C·H()d (Fig. 13). With this equation, the spontaneous preference of the broader of two double stripes can be explained presuming lateral interactions between the components of the patterns (Fig. 8, 10). The strength x _i ^* of this lateral interaction depends on the width of the double stripes. The greater , the smaller is x _i ^* . x _i ^* is a pattern-specific value (Table 1, 2).Supported by the Deutsche Forschungsgemeinschaft, Ho 664/2     

Possible role of Ab b gene in mouse resistance to EL4 metastases

S (survivor) mutants were produced in mice for genetic analysis of host resistance to metastatic cancer. S-mutants S-27 and S-31 resist transplantation of lymphoma EL4 of parental C57BL/6J (B6) mice while they accept parental skin grafts. Mutant S-27 also resists formation of spontaneous metastases from intradermally growing EL4 tumor into lymp nodes; mutant S-31 is highly susceptible to EL4 metastases. Another mutant. H-2 ^bm26 (bm26), resists EL4 and rejects B6 skin grafts. Major histocompatibility complex (MHC) class I and class II gene expression was compared in these mutants and normal B6 mice. All three mutants tested, S-27, S-31, and bm26, expressed a low amount of K ^b mRNA in organspecific fashion. Mutant bm26 and S-31 expressed a low amount of Ab ^b mRNA and of A^b antigen on their spleen cells. Some oligonucleotide probes designed to hybridize to the second exon of the clss II MHC gene Ab ^b did not hybridize with DNA from all three mutants. These findings suggest extensive sequence alternations in the Ab ^b gene in mutants S-27, S-31, and bm26; they also suggest a major role of MHC in the control of host resistance to spontaneous metastases of the EL4 tumor. Address correspondence and offprint request to: I. K.. Egorov.     

Purification and characterization of two intracellular β-glucosidases from the Neurospora crassa mutant cell-1

Two intracellular -glucosidases (E.C. 3.2.1.21) were purified from the filamentous fungus Neurospora crassa, mutant cell-1 (FGSC no. 4335) and characterized. The extent of purification were 2.55- and 28.89-fold for -glucosidase A and -glucosidase B, respectively. -Glucosidase A was a dimeric protein, and B a monomeric protein, with molecular masses of 178 and 106 kDa, respectively. Both isoenzymes were glycoproteins with relatively high carbohydrate contents (-glucosidase A, 29.2%; -glucosidase B, 34.2%). The isoelectric points determined by IEF were 6.27 and 4.72, respectively. pH optima for activity were determined to be 5.0 and 5.5, and temperature optima to be 55 and 60 °C, for -glucosidases A and B, respectively. Both purified -glucosidases. especially -glucosidase B, showed relatively high stability against pH and temperature. Both enzymes were stable in the pH range of 5.0–9.0. The activities were completely retained up to 48 h at temperatures below 40 °C. At higher temperatures, enzymes were relatively unstable and lost their activities at 60 °C after 24 h. Both -glucosidases were highly activated by CuCl₂, and inhibited by SnCl₂ and KMnO₄. Hg²⁺ and Ag⁺ also inhibited severely -glucosidase B. The K _m and V _max values of the isoenzymes against cellobiose as substrate were 1.50 mM and 12.2mol min^–1 mg^–1 for -glucosidase A and 2.76 mM and 143.5 mol min^–1 mg^–1 for -glucosidase B.     

Influence of sarmesin on some dopamine-related types of behaviour

Summary The present study was undertaken to investigate the effects of sarmesin, an analogue of [Sar¹] angiotensin II (ANG II) where the tyrosine hydroxyl group in position 4 is methylated, on dopamine (DA)-related paradigms: locomotor and exploratory behaviour as well as apomorphine (3 mg/kg, ip)-induced stereotypy in rats. Sarmesin (0.5 and 1 g, icv) significantly decreased ambulation and rearing movements, and blocked the inhibitory effect of ANG II (0.1 g) on both types of activity. Sarmesin induced biphasic effects on apomorphine-induced stereotypy depending on the dose increase (0.5 and 5 g, icv) and decrease (10 g). Moreover, sarmesin (5 g) blocked the inhibitory effect of ANG II (2 g, icv) on apomorphine stereotypy. Taken together, these results suggest that sarmesin might interact with AT₁ and AT₂ receptor subtypes. The results further confirm the statement for ANG II-DA interaction in brain structures involved in these types of behaviour.     

High density fed-batch cultures for hybridoma cells performed with the aid of a kinetic model

Hybridoma fed-batch cultures with either standard medium as feed or concentrated medium as feed and removal of toxic metabolites through dialysis were performed by using model calculations for a priori determination of process parameters. In a first step a kinetic model for specific growth and death rate, respectively as well as for substrate uptake and metabolite production rates was formulated. In a bed-batch culture with standard medium as feed the appropriate time for start of the feeding pump and the increase of feed rate were determined a priori. The glutamine concentration was controlled at 0.04 mmoll^–1. A priori calculation and course of the culture coincided rather well. A cell concentration of 3.2×10⁶ cells ml^–1, a MAb-concentration of 54 mg _MAb l^–1 and a MAb-time-space-yield of 0.53 mg _MAb l^–1h^–1 were obtained.For further increase of the efficiency a high density fed-batch process was developed, where concentrated medium is fed to the cells and the accumulating toxic low molecular weight metabolites are removed through a dialysis membrane into a dialyizng fluid. In a membrane dialysis reactor consisting of a culture chamber and a dialyzing chamber, which are separated by a cylindrical dialysis membrane, again model calculations were used to determine feed rate and exchange rate of dialyzing fluid. A viable cell density of 1.2×10⁷ cells ml^–1 and a MAb concentration of 425 mg l^–1 were reached in a culture with stepwise feeding of 10 x concentrated medium and exchange of dialyzing fluid for removal of low molecular metabolites. The course of the culture could be predicted a priori rather well. The MAb-time-space-yield was 2.47 mg _MAb l^–1h^–1, appr. 5 times higher compared to fed-batch cultures with standard medium as feed.List of Symbols A membrane area m² - c _i substrate or product concentration in culture chamber mmoll^–1 - c _a substrate or product concentration in dialyzing chamber mmoll^–1 - c _0i substrate or product concentration in the feed of culture chamber mmoll^–1 - c _0a substrate or product concentration in the feed of dialyzing chamber mmoll^–1 - c _Gln glutamine concentration mmoll^–1 - c _Amm ammonia concentration mmoll^–1 - c _MAb MAb concentration mmoll^–1 - D _a dilution rate in dialyzing chamber d^–1 - F _i feed rate during fed-batch to the culture chamber mlh^–1 - V _a volume of dialyzing chamber l - V _i volume of culture chamber l - P membrane permeability coefficient cm min^–1 - q specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q _Gln spec. glutamine uptake rate mmol cell^–1 h^–1 - q _MAb spec. MAb production rate mmol cell^–1 h^–1 - t time h - X _v viable cell concentration cells ml^–1 - _MAb MAb-time-space-yield mgl^–1 h^–1 - specific growth rate h^–1 - _d specific death rate h^–1 Financial support from the Volkswagen-Stiftung, Germany, grand nr. I/69 359 is gratefully acknowledged.The concentrated medium was kindly provided by SERVA, Heidelberg, Germany. The hybridoma cell line was donated by Prof. fil. dr. Volker Kasche, Technische Universität Hamburg-Harburg, Germany.We express our special thanks to Andreas Schütt, Ralf Gassner, Katja Herbers and Thomas Schäfer for their help in this project.     

Age and Growth of the Whiskery Shark, Furgaleus macki, from Southwestern Australia

Age and growth of the whiskery shark, Furgaleus macki, from southwestern Australia were examined using vertebral ageing and tag-recapture data. The readability of bands on the vertebral centra varied markedly between individuals. Four readers were used to make band counts, with the most experienced reader having the lowest index of average percent error and the highest level of agreement with final counts. Marginal increment analysis indicated that opaque bands form in January. With parturition occurring from August to October, size data suggests that the first band is probably formed 15–17 months after birth. The age at maturity was estimated to be 4.5 years for males, and 6.5 years for females. The oldest male was 10.5 years, and oldest female was 11.5 years. Von Bertalanffy growth parameters for males were L =121.5cm fork length, K=0.423 year^–1, t ₀=–0.472 years, were L =120.7cm fork length, K=0.369 year^–1, t ₀=–0.544 years for females, and were L =118.1cm fork length, K=0.420 year^–1, t ₀=–0.491 years for combined sexes. Data from a tag recapture study were analysed using a maximum likelihood method to verify the estimates of growth parameters from vertebral ageing. Von Bertalanffy growth parameters from the tag recapture study were L =128.2cm fork length, K=0.288 year^–1, t ₀=–0.654 years. The two methods of estimating growth parameters produced similar results, with rapid growth until approximately 5 years of age, after which there was little increase in length.     

Isolation of a rat immune response gene identical to an alleged mouse A class II β-chain pseudogene

A human HLA-DQ -chain cDNA was used as a probe to identify and isolate a rat major histocompatibility antigen -chain gene from a genomic library constructed in the vector Charon 28 using Wistar rat DNA (RT1 ^u). The isolated exon of the rat gene (RT1.B ₂) encoding a -chain second domain was found to share 93% nucleotide homology with a mouse A ₂ exon. Although the genomic organization of this gene is consistent with the hypothesis that it represents a pseudogene, the remarkable preservation of a specific sequence favors the view that this class II antigen -chain gene has retained its coding function.     

Polymorphism of human liver alcohol dehydrogenase: Identification of ADH2 2-1 and ADH2 2-2 phenotypes in the Japanese by isoelectric focusing

Liver homogenate-supernatants from most Japanese exhibit an atypical pH optimum for ethanol oxidation at pH 8.8 instead of 10.5, the typical pH-activity optimum. It has been proposed that atypical livers contain alcohol dehydrogenase isozymes with ₂ subunits while typical livers contain isozymes with ₁ subunits, both produced by the ADH ₂ gene. Because it is difficult to differentiate the atypical ADH₂ 2-2 phenotype from the ADH₂ 2-1 phenotype by starch gel electrophoresis, an agarose isoelectric focusing procedure was developed that clearly separated the atypical Japanese livers into two groups, A1 and A2. The isozymes in A1 and A2 livers were purified. Type A1 livers contained a single isozyme with an atypical pH-rate profile; it was designated ₂₂. Three isozymes were isolated from A2 livers, two of which corresponded to ₁₁ and ₂₂. A third, absent from the typical and the atypical A1 livers, had an intermediate mobility; it was designated ₂₁. Type A1 livers are, therefore, the homozygous ADH₂ 2-2 phenotype, and type A2 livers, the heterozygous ADH₂ 2-1 phenotype. The ADH₂ 2-2 phenotype was found in 53% of 194 Japanese livers, and the ADH₂ 2-1 phenotype, in 31%. Accordingly, the frequency of ADH ₂ ² was 0.68.This study was supported by U.S. Public Health Service Grant AA 02342.     

Methodological problems in evolutionary biology VIII. Biology and culture

Biology cannot accommodate all aspects of culture. Aspects of culture that a biological approach can take into account can be covered by the biological categories of phenotype and environment. There is no need to treat culture as a separate category. Attempts to elaborate biological explanations of cultural variation will meet with success only if biologists expand theories of development, and integrate them in evolutionary biology. The alternative — elaborating the idea of so-called cultural inheritance — makes little sense from a biological point of view.     

The skeletal isotopic composition as an indicator of ecological and physiological plasticity in the coral genus<Emphasis Type="Italic"> Madracis</Emphasis>

Three species of the reef coral genus Madracis display skeletal isotopic characteristics that relate to depth, colony topography, and consequently to coral physiology. The joint interpretation of skeletal ¹³C and ¹⁸O provides information on the ecological plasticity and adaptation to depth of a coral species. Isotopic results are most easily understood in terms of kinetic effects, which reduce both ¹⁸O and ¹³C below isotopic equilibrium values, and metabolic effects, which only influence the skeletal ¹³C. Madracis mirabilis is adapted to depths shallower than 20 m, and shows the greatest range in kinetic effects and the strongest metabolic ¹³C enrichments caused by symbiont photosynthesis. Madracis formosa lives deeper than 40 m, and shows a reduced range of kinetic effects and relatively weak metabolic ¹³C enrichments. Madracis pharensis inhabits depths from 5 to >60 m, and does not attain the strength of kinetic effects of either of the other two species, apparently because it is not quite as well adapted to rapid growth at either extreme.     

N6-(Δ2-Isopentenyl) adenine inhibits the alcohol dehydrogenase activity in cell-free extracts of Zymomonas mobilis

The plant growth substance N⁶-(²-isopentenyl) adenine (i⁶Ade) significantly inhibits the rates of ethanol oxidation and acetaldehyde reduction in vitro by cell-free extracts of Zymomonas mobilis and of an Escherichia coli recombinant strain, containing the Z. mobilis adhB gene. The two-substrate kinetics of ethanol oxidation (forward) is affected by increasing values of dissociation constants for coenzyme and coenzyme —enzyme complexes in the presence of i⁶Ade, whereas the reaction maximum velocity (V _m) remains unchanged and reflects the competitive type of inhibition. Changes of the kinetic constants of acetaldehyde reduction (back) are similar, except the increasing value of V _m and correspond to the CIS (competitive inhibition and stimulation) type of inhibition. The estimated values of inhibition constants of the forward and back reactions are 0.39 ± 0.05 mM and 0.19 ± 0.06 mM, respectively.     

Cloned T cells recognize Trichinella spiralis antigen in association with an Ek βEk α restriction element

A method is described for the production of T-cell lines and clones specific for solubilized Trichinella spiralis antigens. hese T cells are antigen-specific and do not respond to challenge with a third party antigen (lysozyme). The proliferation responses of the cloned T cells are specifically inhibited by anti-I-E but not by anti I-A subregion monoclonal reagents. The inhibition patterns obtained are consistent with cis-gene complementation in B10.K cells involving the E^k -chain and the E^k -chain of the I-E molecule. Inhibition is obtained with an E^k -specific monoclonal antibody (H9-14.8) but not with an A^k -specific monoclonal antibody (10-2.16). Inhibition was also observed with Ia.7-specific (H40-242) or Ia.22-specific (17-3-3) monoclonal antibodies. The inhibition patterns were confirmed by antigen presentation experiments using recombinant inbred mice. Only B 10.K (E^k E^k spleen cells and not B 10.A(5R) (E^b E^k ) or B10.S(9R) (E^s E^k ) spleen cells could effectively present T. spiralis antigens. The role of hybrid Ia molecules in the immune response to T. spiralis is discussed.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

ATP synthesis associated with the conversion of hexachlorocyclohexane related compounds

Clostridium rectum strain S-17 converts -1,2,3,4,5,6-hexachlorocyclohexane (HCH) related compounds to chlorobenzenes. The metabolites from -1,2,3,4,5,6-hexachlorocyclohexene and -1,3,4,5,6-pentachlorocyclohexene are identified as 1,2,4-trichlorobenzene and 1,4-dichlorobenzene, respectively. ATP synthesis, converting these chlorinated compounds, is observed in the cell suspension of C. rectum as indicated by luciferase-luciferin reaction and phosphorylation of ³²P-labeled phosphate. These observation lead to the conclusion that HCH and related compounds serve as artificial electron acceptors of the Stickland reaction, and therefore, the reductive dechlorination is associated with ATP synthesis.Abbreviations HCH -1,2,3,4,5,6-hexachlorocyclohexane - HCCH -1,2,3,4,5,6-hexachlorocyclohexene - PCCH -1,3,4,5,6-pentachlorocyclohexene - TCCH -3,4,5,6-tetrachlorocyclohexene - 1,2,4-TCB 1,2,4-trichlorobenzene - 1,4-DCB 1,4-dichlorobenzene - MCB monochlorobenzene - DTT 1,4-dithiothreitol - IAA monoiodoacetic acid     

Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

T-cell receptor V Tβ genes in natural populations of mice

The composition of 15 V _T gene subfamilies has been examined by Southern hybridization among a broad spectrum of colony bred rat and mouse species extending phylogenetically from Rattus to Mus musculus domesticus. Most mouse species contain a similar content of V _T genes as determined by the number of hybridizing restriction fragment (RF) bands. Furthermore, the extent of restriction fragment length polymorphism (RFLP) appears to be limited. Some V _T gene families, however, are missing from Rattus (VT7, V _T12) and M. shortridgei (V _T9, V _T16). Extension of the V _T survey to a panel of 38 wild-caught mice reveals that nearly a third lack specific hybridization to the V _T5 probe. Previous reports have established that the mouse inbred strains SJL, C57BR, C57L, and SWR lack 50% of their V _T repertoire, including V _T5 (Behlke et al. 1985). This study demonstrates that natural populations of mice also carry a significantly reduced V _T gene repertoire.