2-Chlorobenzoic acid and 2,5-dichlorobenzoic acid metabolism by crude extracts of Pseudomonas sp. CPE2 strain

F. FAVA, F. BALDONI AND L. MARCHETTI. 1996. Crude extracts of Pseudomonas sp. CPE2 strain, which is capable of growing on 2-chlorobenzoic acid (2–CBA) and 2,5–dichlorobenzoic acid (2,5–dCBA) in the absence of other carbon sources, were found to be capable of bioconverting 2–CBA and 2,5–dCBA to catechol and 4–chlorocatechol, respectively, by a reaction requiring molecular oxygen and exogenous NADH. Extracts obtained from 2–CBA-grown cells in the presence of 2–CBA and from 2,5–dCBA-grown cells in the presence of 2,5–dCBA were found to have activities similarly influenced by the assay parameters pH, temperature, and by concentration of oxygen, protein, Fe²⁺, FAD and NADH in the assay medium. In addition, the activity of the two crude extracts in the presence of 2–CBA or 2,5–dCBA was described by very similar Michaelis-Menten kinetic parameters. These observations led to the speculation that a unique broad-spectrum chlorobenzoate 1,2–dioxygenase catalyses the 2–CBA and 2,5–dCBA metabolism both in 2–CBA-and 2,5–dCBA-grown CPE2 cells.     

Two New Species of Isospora from the Desert Skink (Chalchides ocellatus) from the Egyptian Desert

Isospora chalchidis n. sp. and Isospora eimanae n. sp. were isolated from the desert skink, Chalchides ocellatus , order Squamata, from Egypt. Sporulated oocysts of I. chalchidis n. sp. were spheroidal 19 × 19 (18–20.5 × 18–20.5) μm with a 2–layered walled. Oocysts lacked a micropyle and residuum and few polar granules were present. Sporocysts were lemon-shaped 12.2 × 6.5 (9.5–13 × 5–8) μm with a Stieda body and sporocyst residuum but without a substiedal body. Isospora eimanae n. sp. were spheroidal 18.5 × 18.5 (17–19.5 × 17–19.5) μm with a 2–layered wall. There were oocysts without residuum or polar granule, but with small micropyle at 1 end. Sporocysts were ovoid 12 × 8.5(11–13 × 7.5–9) μm with a small Stieda body and sporocyst residuum but without a substiedal body.     

IDENTIFICATION OF MINOR COMPONENTS OF THE SEX PHEROMONE OF YELLOW PEACH MOTH DICHOROCIS PUNCTIFEEALIS GUENÉE AND FIELD TRIALS

Abstract  Along with the major component, (E1–10–hexadecenal (E10–16: Al,), two minor components, (Z)-10-hexadecenal(Z10-l6:Aid) and hexadecanal (16: Ald) were identified as components of the sex pheromone of Dichocrocis punctiferalis Guenee. Analysis of single sex pheromone gland extracts by capillary gas chromatography indicated that the relative ratio of 16:Ald, E10–16:Ald_t and 210–16:Ald was equal to 13. 0:80. 4:6. 6 respectively. Field trails indicated that 210–16: Ald and 16: Aid alone caught no males. The most attractive was a blend containing 16: Ald, E10–16:Ald, and 210–16: Ald at a ratio of 16:100:8, and a two-compound blend of E10–16:Ald and 210–16:Ald at a ratio of 100:8.     

Coefficients of digestibility and nutritional values of geophytes and tubers eaten by southern African mole-rats (Rodentia: Bathyergidae)

Subterranean rodents have high energy requirements when they are excavating their burrows. This study investigates the energy available to, and the efficiency with which it can be extracted by, four species of bathyergid mole-rats fed natural diets ranging from the underground storage organs of geophytes to grass roots and leaves.
The digestibility coefficients of geophytes ranged from 53% for the fibrous tuber of the gemsbok cucumber to 95–7% for corms and bulbs. One species, Bathyergus suillus whose diet consists of over 80% grass, had a digestibility coefficient of 87% on an all grass diet.
All species had similar coefficients of digestibility of > 90% when fed on a uniform diet of sweet potato.
Bulbs and corms had a low fibre content (3–3–4%), high calorific value (15–16kJ/g) and high digestibility coefficients (95–7–96%) and on this diet the mole-rats maintained their body mass. Food of lower digestibility tended to have a high fibre content (8–2–45%) and, with the exception of B. suillus , although the mole-rats consumed a greater quantity of food, they lost mass. The sweet potato had a low fibre content (4–1 %) but was energetically very similar to bulbs and corms (15–5 kJ/g).
Geophytes which have low fibre contents are generally small (1–20 g), whereas geophytes with high fibre contents are much larger (30–2000 g) and often occur in more arid zones.     

Coccidian Parasites (Apicomplexa: Eimeriidae) of Nerodia spp. (Serpentes: Colubridae), with a Description of a New Species of Eimeria

Eimeria conanli n. sp. (Apicomplexa: Eimeriidae) is described from intestinal contents and feces of Nerodia erythrogaster transversa and N harteri harteri from northcentral Texas. Oocysts of the new species are ellipsoid in shape. 17.9 × 13.0(15–21 × 12–15) μm, with a smooth, thin, single-layered wall; shape index 1.4 (1.2–1.5). One to several (usually 2) polar granule(s) and an oocyst residuum are present, but a micropyie is absent. Sporocysts are elongate, 12.9 × 5.2 (13–15 × 5–6) -m, apparently without a true Stieda body structure. Each sporoeyst contains an ellipsoid residuum, 3.9 × 3.2 (3–6 × 2–4) μm, and elongate sporozoites, 11.4 × 2.5 (10–14 × 2–3) μm in situ, each with a spherical or subspherical anterior refractile body and spherical to ellipsoid posterior refractile body. In addition to the new species, oocysts of 4 previously described eimerians from colubrid snakes were found in these hosts.     

Tumor-Promoting Phorbol Esters Promote Melanogenesis and Prevent Expression of the Adrenergic Phenotype in Quail Neural Crest Cells

Tumor-promoting phorbol esters were used to manipulate the in vitro development of neural crest cells. When plated at clonal density in secondary culture, quail neural crest cells from the trunk region gave rise to three types of colonies, pigmented, unpigmented, and mixed. Pigmented colonies consisted exclusively of melanocytes; up to 50% of the unpigmented and mixed colonies contained adrenergic nerve cells which could be identified by a catecholamine-specific histofluorescence method. Addition of potent tumor promoters to the culture medium shortened the doubling time of neural crest cells and altered their morphologic appearance. It also delayed the onset of pigmentation, prevented the expression of the adrenergic phenotype, reduced the number of unpigmented and mixed colonies, and increased the number of pigmented colonies, most likely by directing progenitor cells preferentially to the melanogenic pathway. There was a clear correlation between the ability of phorbol esters to promote skin tumors in mice and their ability to interfere with the in vitro development of quail neural crest cells. The potent promoters 12–0–tetradecanoyl phorbol 13–acetate (TPA) and phorbol 12,13–didecanoate (PDD) were most effective, phorbol 12,13–diacetate (PDA) was considerably less effective, the nonpromoting analogues 4–0–methyl 12–0–tetradecanoyl phorbol 13–acetate (4–0–Me-TPA) and 4α-phorbol 12,13–didecanoate (4α-PDD) and the parent alcohol phorbol (PHR) had little or no effect.     

A medium density microsatellite map of BTA10: reassignment of INRA69

We have developed a genetic map of BTA10 based on 8952 informative meioses for 13 microsatellite markers and the erythrocyte antigen Z. With the exception of OarAE64 , the support for the order of all loci in the map exceeded a LOD > 3·0. The length of the BTA10 genetic map was 87·0 centimorgans (cM). The 14-marker, sex-average map in Kosambi cM was: CSSM38 –8·9- BM1237 –5·2- HH8A –2·6- INRA69 –10·6- TGLA378 –0·8- BM6305 –17·2- TGLA102 –17·9- INRA96 –0·3- CSRM60 –9·2- DIK20 –3·0- EAZ –6·7- CSSM46 –3·7- SRCRSP3 –1·0- OarAE64 with an average interval of 6·70 cM. The microsatellite INRA69 was recently assigned to the pseudoautosomal region of the bovine X chromosome by linkage analysis. However, we found that twopoint support for linkage between INRA69 and 15 X-linked bovine microsatellites was LOD < 0·50 in 529 reciprocal backcross and F₂ fullsib progeny. We performed twopoint analyses of INRA69 against 275 markers distributed throughout the bovine genome and found significant associations with a LOD > 3·0 only between INRA69 and eight BTA10 microsatellite loci. Consequently, we excluded INRA69 from the genetic map of the X chromosome and reassign this microsatellite to BTA10.     

An ER membrane protein,Sop4, facilitates ER export of the yeast plasma membrane [H+]ATPase,Pma1

We have analyzed the mechanism by which Sop4, a novel ER membrane protein, regulates quality control and intracellular transport of Pma1–7, a mutant plasma membrane ATPase. At the restrictive temperature, newly synthesized Pma1–7 is targeted for vacuolar degradation instead of being correctly delivered to the cell surface. Loss of Sop4 at least partially corrects vacuolar mislocalization, allowing Pma1–7 routing to the plasma membrane. Ste2–3 is a mutant pheromone receptor which, like Pma1–7, is defective in targeting to the cell surface, resulting in a mating defect. sop4Δ suppresses the mating defect of ste2–3 cells as well as the growth defect of pma1–7 . Visualization of newly synthesized Pma1–7 in sop4Δ cells by indirect immunofluorescence reveals delayed export from the ER. Similarly, ER export of wild-type Pma1 is delayed in the absence of Sop4 although intracellular transport of Gas1 and CPY is unaffected. These observations suggest a model in which a selective increase in ER residence time for Pma1–7 may allow it to achieve a more favorable conformation for subsequent delivery to the plasma membrane. In support of this model, newly synthesized Pma1–7 is also routed to the plasma membrane upon release from a general block of ER-to-Golgi transport in sec13–1 cells.     

Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K-12 depends on DNA binding site arrangement

The NarL and NarP proteins are homologous response regulators of Escherichia coli that control the expression of several operons in response to nitrate and nitrite. A consensus heptameric NarL DNA-binding sequence has been identified, and previous observations suggest that the NarP protein has a similar sequence specificity. However, some operons are regulated by NarL alone, whereas others are controlled by both NarL and NarP. In this study, DNase I footprinting experiments with the fdnG , nirB and nrfA control regions revealed that NarP only binds to heptamer sequences organized as an inverted repeat with a 2 bp spacing (7–2–7 sites). The NarL protein also binds to these 7–2–7 sites but, unlike NarP, also recognizes heptamers in other arrangements. These results provide an explanation for the regulation of some operons by NarL alone and for the different effects of NarL and NarP at common target operons, such as fdnG and nrfA . To investigate this differential DNA binding further, derivatives of the nrfA control region were constructed in which the spacing of the 7–2–7 heptamers was increased (7– n –7 constructs). Increasing the spacing to four or more basepairs abolished NarP binding and significantly reduced NarL binding. The NarL protein also had a reduced binding affinity for heptamers adjacent to the 7– n –7 heptamer pair, suggesting a decrease in cooperative interactions. In conclusion, we propose that 7–2–7 sites are preferred by both NarL and NarP. NarL can also recognize other binding site arrangements, an ability that appears to be lacking in NarP.     

Description of the Oocysts of Two New Species of Eimeria (Apicomplexa: Eimerüdae) from the Florida Manatee, Trichechus manatus (Sirenia: Trichechidae)

Two new species of Eimeria (Apicomplexa: Eimerüdae) are described from the feces of the Florida manatee, Trichechus manatus latirostrts (Sirenia: Trichechidae). Oocysts of Eimeria manatus n. sp. are spherical to subsphencal, 11.8 × 10.7 (10.5–13.5 × 9.0–13.5) μ m , with a smooth, thin, bilayered wall; shape index (length/width) 1.1 (1.0–1.3). Micropyle and oocyst residuum absent; polar granule(s) usually present. Sporocysts are ovoid, 8.6 × 5.1 (8.0–9.5 × 5.0–5.5) μm, with thin, membrane–like walls and a knoblike Stieda body; shape index 1.7 (1.4–1.8). Sporozoites elongate, each with a large posterior refractile body. The sporocyst residuum consists of a small cluster or row of few to many small granules. Oocysts of Eimeria nodulosa n. sp. are spherical to subspherical, 15.6 × 14.7 (14.5–17.5 × 13.0–16.0) μm, with a distinctly bilayered wall; shape index 1.1 (1.0–1.2). Unsporulated and freshly sporulated oocysts often possess large, knob–like structures on the external surface of the oocyst wall that support a thin membrane or filament. Micropyle, oocyst residuum and polar granule absent. Sporocysts are ovoid, 10.6 × 5.9 (9.5–12.0 × 5.0–6.5) μm, with a smooth, thin wall and knob–like Stieda body; shape index 1.8 (1.5–2.1). Sporozoites granular and elongate, each with a large posterior refractile body. The sporocyst residuum consists of a loose aggregate or scattered mass of moderately sized granules.     

Upp er lethal temperature tolerance of fingerling black crapp ie

Upper lethal temperature tolerance was determined for 26–81 mm, age 0 black crappie Pomoxis nigromaculatus of three size classes using both a rapid transfer and slow heating protocol. Rapid transfer protocols determined 24 LT₅₀ values of 33–8, 35–1 and 31–5° C for size classes with mean total lengths of 30–2, 45–6 and 74–9 mm. A predictive model was generated from the slow heating protocols that relates lethal temperature to acclimation temperature, total length, and condition factor (K) as predictors.     

Micromere Differentiation in the Sea Urchin Embryo: Expression of Primary Mesenchyme Cell Specific Antigen during Development

The temporal and spatial expression of antigen specific for primary mesenchyme cell (PMC) lineage cells during early development of the sea urchins Hemicentrotus pulcherrimus and Stronglyocentrotus nudus was studied with a monoclonal antibody (P4). P4 was produced by a hybridoma cell line prepared by fusion of myeloma cells and spleen cells from a mouse immunized with cultured spicule-forming cells. Immunofluorescence studies demonstrated that P4 antibody reacted strongly with the surfaces of PMC's and spicule-forming cells of both species. Immunoblot analysis showed that P4 antibody reacted with several proteins including those of 140–kDa, 120–kDa, 53-kDa, 43–kDa, and 41–kDa in H. pulcherrimus and with those of 130–kDa, 110–kDa, 51–kDa, and 43–kDa in S. nudus . These proteins appeared sequentially after the hatching blastula stage. Tunicamycin inhibited the expressions of these P4 antigens as well as spicule formation. Two of the P4-reactive antigens, the 140–kDa and 43–kDa proteins, in H. pulcherrimus were synthesized de novo and shown to be identical to micromere differentiation specific proteins. These results suggest that P4 binds to specific molecules that are important in spicule formation in developing sea urchin embryos.     

Amidation of β-Amyloid Peptide Strongly Reduced the Amyloidogenic Activity Without Alteration of the Neurotoxicity

Abstract: β-Amyloid accumulates in cerebral deposits in Alzheimer's disease, so to test the correlation between the neurotoxic and fibrillogenic capacity of β-amyloid, we synthesized a peptide homologous to fragment 25–35 of β-amyloid (β25–35) and amidated at the C-terminus (β25–35-NH₂). As the amidation strongly reduced the amyloidogenic capacity of β25–35, we compared its neurotoxic activity in the amidated (β25–35-NH₂) and nonamidated forms. The viability of primary cultures from fetal rat hippocampus was reduced in a dose-related manner (10–100 µ M ) similarly by β25–35 and β25–35-NH₂, whereas a scrambled peptide, amidated or nonamidated, did not alter the neuronal viability. The neurotoxic activity of β25–35-NH₂ is mediated by apoptosis as demonstrated by morphological and biochemical investigations. Electron microscopy examination of culture media with β25–35 or β25–35-NH₂ incubated with neuronal cells for 7 days confirmed the high level of fibrillogenic activity of β25–35 and the almost total absence of fibrils in the solution with β25–35-NH₂. Furthermore, staining with thioflavine S was used to identify amyloid fibrils, and only the cultures exposed to β25–35 exhibited intense staining associated with neuronal membranes. These data indicate that the neurotoxic activity of the β-amyloid fragment is independent of the aggregated state of the peptide.     

STAGING OF NEWT BLASTULA EMBRYOS AND FIRST APPEARANCE OF PRIMARY MESODERMAL COMPETENCE

Using the swimbladder of the crusian carp ( Carrasius auratus ) as an inductor, the first appearance of mesodermal competence in the presumptive ectoderm of the newt ( Triturus pyrrhogaster ) blastula was investigated. The time course of embryonic development before the gastrula stage was determined by counting the number of surface cells on a 0.25 mm line at the animal pole. Pregastrula embryos with 2–3, 4–5, 6–7 and 7–8 cells roughly correspond to those at 14, 14–12, 8–6 and 4–0 hr before the beginning of gastrulation. Using presumptive ectoderm of the early gastrula stage, 15 min was found to be the minimum time of contact necessary for the realization of induction. The reactivity of the presumptive ectoderm from pregastrula embryos was tested by 30 min contact. Presumptive ectoderm up to the 4–5 cell stage did not react to the inductor. It may become competent within the next 4–8 hr, since the ectoderm from embryos in the 6–7 cell stage was reactive.     

THE LYMPHOCYTES OF CHRONIC LYMPHOCYTIC LEUKEMIA: THEIR PROLIFERATION AND CELL CYCLE KINETICS

Lymphocytes obtained from CLL patients exhibited a delayed and reduced response to PHA when cultured in diffusion chambers. DNA synthesis (8–10 hr) and general time (15–19 hr) of the late-developing CLL blasts were consistent with normal values ( T _s: 8–10 hr; T _c: 14–17 hr). However, the G₂ period of CLL blasts seemed more variable, and their mitotic index during the response at 5–6 days was 30–50% of the values determined for normal blasts during their peak response at 2–3 days.     

Production of metabolic and waste products by intensively farmed rainbow trout, Salmo gaivdnevi Richardson

The pollution production rate as measured by the increase in the amounts of ammonia, phosphate, nitrate, urea, and faeces in an intensive fish farm is described and is related to the amount of food fed per day or the biomass weight. Pollution production varied with fish size. Main pollutants produced per kg food fed per day were ammonia 31–37 g; phosphate 5.2–5.9 g; nitrate 9–15 g and suspended solids 40–9O g. Expressed as g kg⁻¹ fish produced per day ammonia ranged from 0.3–0.8 g; phosphate 0.067–0.17 g; nitrate 0.13–0.21 g and suspended solids 0.80–0.94 g. These rates differ from those reported in previous studies and these differences may be attributed to the design of the Shearwater farming system which involves self cleaning, intensively stocked tanks, a system which ultimately gives a more accurate assessment of pollution rates.     

Production of monoclonal antibodies against Clostridium botulinum type E derivative toxin

Abstract Five monoclonal antibodies (MCA; E–8–2, 9–1, 11–2, 12–4, and 13–1) against Clostridium botulinum type E derivative toxin were prepared. Their ELISA titers were higher than or equivalent to that of conventional polyclonal antibody. Three of them (E-8–2, 12–4, and 13–1) possessed the neutralizing activity comparable to that of polyclonal antibody. The results of binding-competition experiments indicated that the monoclonal antibodies bound to different sites on the type E toxin molecule. Immunoblotting analyses demonstrated that E-8–2, 9–1, and 11–2 react to fragment I (heavy chain) of the toxin. By use of these monoclonal antibodies, it may be possible to scrutinize the structure-function relationship of botulinum toxins and cross reactions between type E and F toxins.     

A biological analysis of eel catches, Anguilla anguilla L., from the lagoons of Lesina and Varano, Italy

A study of eel catches from Lesina (444 specimens) and Varano lagoons (325 specimens), in southern Adriatic, Italy, was made. Male silver eels in Lesina ranged from 33.4–51.5 cm in length, with a mean of 42.6 cm; from 50–240 g in weight, with a mean of 141 g and were 1.5–6.5 years old with a mean of 2.5 years. The average length of male silver eels in Varano lagoon was 40.5 cm (range 31–48.5 cm); the average weight was 122 g (range 80–220 g)and a mean age of 2.6 years (range 1.5–7.5 years).
The females are bigger, heavier and older than the males with, in Lesina, a mean length of 61 cm (range 50.9–74.3 cm), a mean weight of 438 g (range 240–730 g) and a mean age of 3.4 years (range 1.5–6.5). The average length of Varano female silver eels was 58 cm (range 50.8–72.5 cm), and the average weight was 383 g (range 225–840 g). They were 1.5–7.5 years old, with an average of 3.8 years. Female silver eels were only 20% of the population at Lesina and 10% at Varano.
In comparison with the silver eel populations of the North Adriatic lagoons, the North Sea or the Atlantic Ocean, the silver eels of Lesina and Varano show a greater growth rate, are younger and have a sex ratio in favour of the males.
The water temperature, higher than in other countries, could be an important factor affecting the differences in age and growth rates between Lesina and Varano silver eels and those of other waters.     

Isolation and Characterization of Pseudomonas syringae subsp. savastanoi Mutants Defective in Hypersensitive Response Elicitation and Pathogenicity

Olive strain ITM317 of Pseudomonas syringae subsp. savastanoi , the causal agent of 'Olive and Oleander knot disease' was mutagenized by random transposon (Tn5) insertion. Of the 1 400 transconjugants, four were altered in their ability to induce a hypersensitive reaction (HR) on tobacco; Southern blot analysis showed that a single copy of the Tn5 element was present in their chromosomes. In particular, mutants ITM317–69, ITM317–1010 and ITM317–1194 did not elicit HR whereas mutant ITM317–916 induced a variable response. When assayed for pathogenicity on olive, mutants ITM317–916 and ITM317–1010 induced knots comparable both in size and morphology to those caused by the parental strain. Prototrophic mutant ITM317–1194, still able to produce indole-3-acetic acid and cytokinins, did not cause any knot formation on olive; furthermore, it was unable to multiply in host tissue. Auxotrophic mutant ITM317–69 caused the formation of smaller-sized knots and its prototrophic revertant fully regained the parental phenotypes, suggesting that a single Tn5 insertion had a pleiotropic effect on the mutated phenotypes. Tn5-containing Eco RI fragments from mutants ITM317–69, ITM317–916, ITM317–1010 and ITM317–1194 were cloned into the plasmid vector pBR322. Hybridization of these clones with the hrp gene cluster of P. s. pv. syringae strain 61 was not detected. These results suggest that genes different from those of the above gene cluster might be involved in the interaction of P. s. subsp. savastanoi with olive and with the non-host plant tobacco.     

Sex ratio in the spider Pityohyphantes phrygianus affected by winter severity

The sex ratio of subadult Pityohyphantes phrygianus was recorded before and after five winters in a natural population in coniferous forest in south-west Sweden. In autumn, the proportion of males was on average 33–8% (range 28–7–40–1) and the proportion of females was 66–2% (59–9–71–3). In each of the winters 1981 82, 1982–83, 1984–5, the proportion of males decreased significantly. The proportion of males decreased more the lower the February mean temperature. Field experiments showed that low ambient temperature in winter caused high mortality among spiders. Experimental data also suggest that males are more vulnerable to low winter temperatures than are females. Indirect evidence indicates that neither predation pressure nor starvation alone are likely to cause the observed changes in sex ratio in the three winters. However, the combined effect of sex differences in predation by birds and cold-induced mortality may explain why males disappear faster than females in certain winters.