Biochemical identification of the bovine blood group M' antigen as a major histocompatibility complex class I-like molecule

Absorption and elution experiments showed that it was impossible to separate antibodies against blood group factor M' from antibodies against bovine lymphocyte antigen (BoLA) A16 in an antiserum showing haemolytic activity against M' as well as lymphocytotoxic activity against BoLA-A16. To elucidate the structural relationship between BoLA-A16 and blood group antigen M', immunoprecipitation experiments on red and white cell lysates isolated from M'-A16 positive and negative cattle were carried out. These results showed that M_r 44 000 and M_r 12000 polypeptides can be precipitated from both red and white cells isolated from M'-A16 positive animals, whereas no bands were seen in M'-A16 negative animals in precipitations with the same antibody. Precipitation with a crossreacting human β₂-microglobulin (β₂-m) specific antibody confirmed a class-I-like structure associated with β₂-m on M' positive red cells and the absence of such a structure on M' negative red cells. Sequential precipitations gave analogous results. Proteolytic degradation by papain and V8 protease did not reveal any substantial difference between red and white M'-A16 positive cells, but a slight difference in the pI of the immunoprecipitable components of red and white cells was observed. All together, this indicates that either the blood group antigen M' is the BoLA-A16 class I antigen or M' and BoLA-A16 are two different class I polypeptides with the same relative mass, sharing identical epitopes and both associated with β₂-m. Comparable results were obtained with M₁ and BoLA-A24.     

Flow cytometric correlation of the c-myc oncoprotein and cell cycle kinetics of HL60 leukaemia during induced maturation with cytosine arabinoside and dimethylsulphoxide

Abstract The c-myc oncogene codes for a DNA binding protein that functions in a cell cycle-related manner. A useful model for studying the relationship of c-myc expression with cell cycle kinetics is the HL60 cell line. HL60 cells constitutively express high levels of c-myc mRNA; however, the level can be down-regulated as the cells are induced to differentiate. We have developed a flow cytometric assay for correlating c-myc oncoprotein levels with DNA content. C-myc oncoprotein levels were additionally correlated with c-myc mRNA levels as determined by slot blot hybridization. Dimethylsulphoxide (DMSO) and cytosine arabinoside were used to induce granulocytic and monocytic maturation respectively. Treatment of HL60 cells with DMSO leads to an increase in the per cent of cells in G₁/G₀ and a decrease in mean c-myc mRNA and oncoprotein levels. The cells with G₁ DNA content show the greatest decrease in c-myc protein. ARA-c treatment of HL60 cells leads to a slowing and an accumulation of cells in S phase with a moderate decrease in mean mRNA and only a slight decrease in mean c-myc protein levels. These data support the hypothesis that c-myc is involved in the switch from G₁ to G₀.     

Allozyme, mitochondrial-DNA, and morphometrie variability indicate cryptic species of anchovy (Engraulis encrasicolus)

Previous surveys of population structure in the Atlantic-Mediterranean anchovy Engraulis encrasicolus L. have reported heterogeneity in morphology, allozyme frequencies, and mitochondrial DNA haplotype frequencies at a regional scale. In particular, two stocks of anchovy have been detected in the Adriatic Sea. In this paper, the available data is reviewed with the aim to relate genetic variation to geography at the widest possible geographical scale, for investigating the evolutionary mechanisms underlying stock structure in anchovy. Correspondence analysis of allozyme frequencies (24 samples, three polymorphic loci) compiled from the literature indicates three distinct entities in the Mediterranean Sea. Open-sea or oceanic anchovy populations are genetically different from inshore-water populations within a region (Nei's ^ G _ST = 0.035–0.067), while broadscale geographical variation is weak for each of these two habitat-specific forms (^ G _ST = 0.005–0.006). Mitochondrial-DNA haplotype frequencies support the distinction between an inshore form and an oceanic form (^ G _ST = 0.067–0.107), with virtually no genetic differences among oceanic populations across the Gulf of Biscay, the western Mediterranean and the Ionian Sea (^ G _ST = −0.001). If natural selection on marker loci is unimportant, these results indicate the occurrence of two parapatric, genetically distinct, habitat-specific forms that are widely distributed throughout the Mediterranean Sea. Persistent allele and haplotype-frequency differences between these forms indicate reproductive isolation and the presence of an E. encrasicolus species complex in the Mediterranean. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society 75 : 261–269.     

Rapid Degeneration of Cultured Human Brain Pericytes by Amyloid β Protein

Abstract: Amyloid β protein (Aβ) deposition in the cerebral arterial and capillary walls is one of the major characteristics of brains from patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Vascular Aβ deposition is accompanied by degeneration of smooth muscle cells and pericytes. In this study we found that Aβ_1–40 carrying the "Dutch" mutation (HCHWA-D Aβ_1–40) as well as wild-type Aβ_1–42 induced degeneration of cultured human brain pericytes and human leptomeningeal smooth muscle cells, whereas wild-type Aβ_1–40 and HCHWA-D Aβ_1–42 were inactive. Cultured brain pericytes appeared to be much more vulnerable to Aβ-induced degeneration than leptomeningeal smooth muscle cells, because in brain pericyte cultures cell viability already decreased after 2 days of exposure to HCHWA-D Aβ_1–40, whereas in leptomeningeal smooth muscle cell cultures cell death was prominent only after 4–5 days. Moreover, leptomeningeal smooth muscle cell cultures were better able to recover than brain pericyte cultures after short-term treatment with HCHWA-D Aβ_1–40. Degeneration of either cell type was preceded by an increased production of cellular amyloid precursor protein. Both cell death and amyloid precursor protein production could be inhibited by the amyloid-binding dye Congo red, suggesting that fibril assembly of Aβ is crucial for initiating its destructive effects. These data imply an important role for Aβ in inducing perivascular cell pathology as observed in the cerebral vasculature of patients with Alzheimer's disease or HCHWA-D.     

Occurrence of aminopropylcadaverine and its aminopropyl derivatives aminopentylnorspermidine and N, N' -bis(3-aminopropyl)cadaverine in Halococcus acetoinfaciens

Abstract Besides putrescine, cadaverine, spermidine, spermine and thermospermine, three novel polyamines were detected in a slightly halophilic eubacterium Halococcus acetoinfaciens (IAM 12094, ATCC 25861). These novel polyamines were found to be N -3-aminopropylcadaverine [NH₂(CH₂)₃NH(CH₂)₅NH₂] and its aminopropyl derivatives: aminopentylnorspermidine [NH₂(CH₂)₃NH(CH₂)₃NH(CH₂)₅NH₂] and N , N ' -bis(3-aminoprophyl)cadaverine [NH₂(CH₂)₃ NH(CH₂)₅NH(CH₂)₃NH₂]. Aminopropylcadaverine was also detected in two other species, Halococcus agglomeratus (IAM 12095, ATCC 25862) and Halococcus nondenitrificans (IAM 12096, ATCC 25863).     

Structure and Functional Characterization of a Novel Human Low-Voltage Activated Calcium Channel

Abstract : We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca²⁺ channel α₁ subunit, α_1H, from a human medullary thyroid carcinoma cell line. The α_1H subunit is structurally similar to previously described α₁ subunits. Northern blot analysis indicates that α_1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba²⁺ currents recorded from human embryonic kidney 293 cells transiently expressing α_1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (τ = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing α_1H. Singlechannel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of ~9 pS. These channels are blocked by Ni²⁺ (IC₅₀ = 6.6 μ M ) and the T-type channel antagonists mibefradil (~50% block at 1 μ M ) and amiloride (IC₅₀ = 167 μ M ). Thus, α_1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca²⁺ channels.     

Measurement of c-myc protein content and cell cycle kinetics of normal and spontaneously transformed murine mastocytes by bivariate flow cytometry

Abstract. Progressive in vitro culturing of interleukin-3 (IL-3) dependent normal murine mastocytes (PB-3) resulted in a variant cell line (PB-1) able to grow without exogenous IL-3 and which was tumorogenic in syngenic mice. Bivariate flow cytometry was used to evaluate the c-myc protein and DNA content of PB-3 and PB-1 cells. The c-myc protein was detected by specific monoclonal antibodies. Kinetic characteristics of PB-3 and PB-1 cell lines, namely, the duration of the G¹, S and G₂+ M cell cycle phases were also evaluated using the bromodeoxyuridine (BrdU) pulse-chase method and BrdU/DNA flow cytometry. Levels of c-myc protein in PB-1 cells were about twofold higher than those of PB-3 cells in all cell cycle phases. Mean duration of the cell cycle ( T _c) was 15-3 h for PB-3 cells and 12-4 h for PB-1 cells. Shortening in T _c for the transformed cells was due to a decrease of nearly 30% in mean duration of the G ₁ phase (from 8 h to 5.7 h). No significant differences were found in the duration of the S and G₂+ M phases. These results indicate that acquired IL-3 independency in vitro and tumorogenicity of PB-1 cells were accompanied by a doubling of c-myc protein level and by a parallel shortening, or bypass, of the regulatory events within the G¹ phase of the cell cycle.     

Pathologic Amyloid β-Protein Cell Surface Fibril Assembly on Cultured Human Cerebrovascular Smooth Muscle Cells

Abstract: Cerebrovascular amyloid β-protein (Aβ) deposition is a key pathological feature of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Aβ_1–40 containing the E22Q HCHWA-D mutation, but not wild-type Aβ_1–40, potently induces several pathologic responses in cultured human cerebrovascular smooth muscle cells, including cellular degeneration and a robust increase in the levels of cellular Aβ precursor. In the present study, we show by several quantitative criteria, including thioflavin T fluorescence binding, circular dichroism spectroscopy, and transmission electron microscopic analysis, that at a concentration of 25 µ M neither HCHWA-D Aβ_1–40 nor wild-type Aβ_1–40 appreciably assembles into β-pleated sheet-containing fibrils in solution over a 6-day incubation period. In contrast, at the same concentrations, HCHWA-D Aβ_1–40, but not wild-type Aβ_1–40, selectively binds and assembles into abundant fibrils on the surfaces of cultured human cerebrovascular smooth muscle cells. The simultaneous addition of an equimolar concentration of the dye Congo red prevents the cell surface fibril assembly of HCHWA-D Aβ_1–40. Moreover, Congo red effectively blocks the key pathologic responses induced by HCHWA-D Aβ_1–40 in these cells. The present findings suggest that the surface of human cerebrovascular smooth muscle cells may selectively orchestrate the assembly of pathogenic Aβ fibrils and that cell surface Aβ fibril formation plays an important role in causing the pathologic responses in these cells.     

Oxygen uptake in coprophilous beetles (Aphodius, Geotrupes, Sphaeridium) at low oxygen and high carbon dioxide concentrations

Abstract. The rate of O₂ consumption was measured in five coprophilous beetle species (common in Denmark) at O₂ concentrations from 1–21%. With the exception of the mainly soil-living Geotrupes spiniger (Marsham) (Geotrupidae), these beetles are probably exposed to severe hypoxia in fresh cattle pats. Aphodius fossor (Linnaeus), A. contaminatus (Herbst) (Aphodiidae) and Sphaeridium lunatum Fabricius (Hydrophilidae) maintained normal movements and a normal rate of 0₂ uptake (for at least 30 min) at only 1% O₂. There is no evidence, therefore, that the beetles switch to anaerobic metabolism under these conditions. This ability to regulate respiration, and hence to extract 0₂ at very low concentrations, is exceptional even among terrestrial arthropods living in soil or other potentially hypoxic substrates. In A. rufipes (Linnaeus), respiration declined at ambient concentrations below 2% O₂, and in G. spiniger the ability to regulate respiration seemed to fail at even higher concentrations. In four of the species (G. spiniger was not tested), about 11% CO₂ (the level in a dung pat at 2% O₂) did not affect the O₂ uptake at 2% O₂.     

Scl1-dependent internalization of group A Streptococcus via direct interactions with the alpha2beta(1) integrin enhances pathogen survival and re-emergence

The molecular pathogenesis of infections caused by group A Streptococcus (GAS) is not fully understood. We recently reported that a recombinant protein derived from the collagen-like surface protein, Scl1, bound to the human collagen receptor, integrin α₂β₁. Here, we investigate whether the same Scl1 variant expressed by GAS cells interacts with the integrin α₂β₁ and affects the biological outcome of host–pathogen interactions. We demonstrate that GAS adherence and internalization involve direct interactions between surface expressed Scl1 and the α₂β₁ integrin, because (i) both adherence and internalization of the scl1- inactivated mutant were significantly decreased, and were restored by in-trans complementation of Scl1 expression, (ii) GAS internalization was reduced by pre-treatment of HEp-2 cells with anti-α₂ integrin-subunit antibody and type I collagen, (iii) recombinant α₂-I domain bound the wild-type GAS cells and (iv) internalization of wild-type cells was significantly increased in C2C12 cells expressing the α₂β₁ integrin as the only collagen-binding integrin. Next, we determined that internalized GAS re-emerges from epithelial cells into the extracellular environment. Taken together, our data describe a new molecular mechanism used by GAS involving the direct interaction between Scl1 and integrins, which increases the overall capability of the pathogen to survive and re-emerge.     

Response properties of CO2-sensitive receptors in tsetse flies (Diptera: Glossina Palpalis)

Abstract. Extracellular single cell recordings of CO₂-sensitive receptors in tsetse flies revealed a steep dose response over a range of stimuli of two to three orders of magnitude and a maximum response of approximately 70 impulses/s after exposure to a high, but naturally feasible, C0₂ concentration of 5%. These receptor neurones are slightly sensitive to C0₂ levels occurring in air (-0.03%); the sensitivity to CO² above that level may be used to locate potential hosts. The C0₂-sensitive neurones did not respond to some other biologically relevant odours such as octenol, butanone or p-cresol; however, other receptor cells, some in the same sensillum, are sensitive to some of these odours.
A striking feature of the C0₂ receptors is that they appear not to adapt in their response frequencies. The spike numbers of the phasic-tonic response remain constant in the tonic portion during continuous (or repetitively pulsed) long-term stimulation (1 min). This unusual physiological ability would allow continuous monitoring of C0₂ values as well as detecting potential hosts that exhale CO₂. These electrophysiological results are compared to the behavioural findings in tsetse flies and to the physiological data on C0₂ receptors of other, non-bloodsucking insects.     

A qRT-PCR-based method for the measurement of rrn operon copy number

Aim:  To develop a convenient and accurate method for estimating the rrn operon copy number ( Y _rrn ) in cells of pure prokaryotic cultures based on quantitative real-time polymerase chain reaction (qRT-PCR).
Methods & Results:  Using Escherichia coli, the Y _rrn of which is known to be 7, as a reference, the rrn concentrations of target species and E. coli in sample solutions were measured based on their respective threshold cycle numbers ( C _t ), whereas the cell concentrations of both species were measured by microscopic counting after staining. The Y _rrn of the target species was then calculated from the initial cell concentrations and the rrn concentrations of the target species and E. coli . Using this method, the Y _rrn values of four species, i.e. Xanthomonas campestris , Staphylococcus aureus , Aeromonas hydrophila and Pseudomonas fluorescens , were estimated as 1·80, 4·73, 8·58 and 5·13, respectively, comparable to their respective known values of 2, 5, 10, and 5, resulting in an average deviation of 8%.
Conclusions:  The whole cell qRT-PCR based methods were convenient, accurate and reproducible in quantification of rrn copy number of prokaryotic cells.
Significance and Impact of the Study:  qTR-PCR is a fast and reliable DNA quantification approach. Compared with previous qTR-PCR based methods measuring rrn copy number, the present method avoided the prerequisite for the information on genome size and GC content of target bacteria or a gene with known copy number, thus should be more widely applicable.     

Inheritance of Anthracnose Resistance in the Common Bean Differential Cultivar G 2333 and Identification of a New Molecular Marker Linked to the Co-42 gene

The inheritance of anthracnose resistance of the common bean ( Phaseolus vulgaris L.) differential cultivar G 2333 to Colletotrichum lindemuthianum races 73 and 89 was studied in crosses with the susceptible cultivar Rudá. The segregation ratios of 15 : 1 in the F₂ and 3 : 1 in the backcrosses to Rudá indicate that for each of the races tested there are two independent resistance loci in G 2333. A random amplified polymorphic DNA (RAPD) molecular marker (OPH18_1200C) linked in resistance to race 73 was identified in a BC₃F_2:3 population derived from crosses between Rudá and G 2333. A RAPD molecular marker OPAS13_950C, previously identified as linked to gene Co-4² , was also amplified in this population. Co-segregation analyses showed that these two markers are located at 5.6 (OPH18_1200C) and 11.2 (OPAS13_950C) cM of the Co-4² gene. These markers were not present in BC₁F_2:3 plants resistant to race 89 indicating that this population carries a different resistance gene. DNA amplification of BC₁F_2:3 plants with RAPD molecular marker OPAB_450C, previously identified as linked to gene Co-5 , indicated that this gene is present in this population.     

Compensatory growth of juvenile brown flounder Paralichthys olivaceus (Temminck & Schlegel) following thermal manipulation

The compensatory growth of juvenile brown flounder Paralichthys olivaceus (body mass c. 12 g) following different thermal exposure was investigated. Fish were exposed to one of the five temperatures: 8·5 ( T _8·5), 13·0 ( T _13·0), 17·5 ( T _17·5), 22·0 ( T _22·0) and 26·5° C ( T _26·5) for 10 days and fish grew best at 22·0° C. Then the water temperature in all treatments was equably adjusted to 22·0° C over 3 days. At the end of the following 30 days after temperature adjustment, there were no significant differences between body masses of fish in the different treatments (wet body mass at the end of the experiment ranged from 22·13 to 24·56 g). Results indicated that the juvenile P. olivaceus achieved complete compensatory growth. Analysis of the dynamics of the feeding rates and feed conversion efficiencies indicated that compensatory growth of the fish experienced low temperature ( T _8·5, T _13·5 and T _17·5) or high temperature ( T _26·5) exposure was mainly dependent on increasing feed intake (hyperphagia) and possibly by improvement in feed conversion efficiency. The moisture content was not affected by different temperature exposure significantly. The lipid and energy content of juvenile P. olivaceus in T _8·5, however, were significantly lower than other treatment. Results of the current study indicate that a short period of low or high temperature exposure may not affect annual growth, but may affect lipid and energy deposition.     

Streptococcus pyogenes grown under sublethal concentrations of penicillin G accumulates close to the septum pentapeptide subunits of peptidoglycan

Abstract The accumulation of non-crosslinked pentapeptide subunits of peptidoglycan was localized close to the septum in cells of Streptococcus pyogenes , grown under sublethal concentrations of penicillin G. Pentapeptide subunits were detected in the cell wall by the indirect immunoferritin technique, employing monospecific antibodies to the sequence R- d -Ala- d -Ala-OH. Antibodies of this specificity were obtained by immunization with synthetic immunogen albumin-(CH₂CO-Gly- l -Ala₂- d -Ala- d -Ala-OH)₃₉.     

A dual block to cell cycle progression in HL60 cells exposed to analogues of vitamin D,

Abstract. The physiologically active form of vitamin D₃, 1,25-dihydroxy-vitamin D₃, (1,25(OH)₂, D₃), induces differentiation of several types of myeloid leukaemia cells. The acquisition of monocyte-like phenotype is accompanied by slower progression through the cell cycle, and G₁, block has been reported to be the basis of this effect. It is shown here that human promyelocytic leukaemia HL60 cells treated with analogues of vitamin D₃, which are potent inducers of monocytic differentiation, have an additional cell cycle block. Exposure to 10-⁷m 1,25(OH)₂, D₃, or 1,25-(OH)₂,-16-ene-D₃ resulted in monocytic differentiation and the expected G₁, block evident at approximately 48 h in a rapidly differentiating variant of HL60 cells (HL60-G), and at 96 h in the more slowly differentiating HL60-240 cells. In addition, a G₂,+M block was noted at approximately 72 h in HL60-G and HL60-240 cells. Exposure to vitamin D₃, analogues also markedly increased the number of dikaryons, suggesting that cytokinesis was impaired more than karyokinesis. Treatment with a third analogue 25-hydroxy-16,23-diene-D₃, produced little differentiation and had minimal effects on the cell cycle parameters. These findings indicate that vitamin D₃, analogues regulate cell proliferation by control of the transition of G₁, and G₂,+M phases, reminiscent of the cdc2/CDK2 type of cell cycle control.     

Cell wall modifications of bean (Phaseolus vulgaris) cell suspensions during habituation and dehabituation to dichlobenil

Bean ( Phaseolus vulgaris L.) cell suspensions were adapted for growth in 12 µ M dichlobenil (2,6-dichlorobenzonitrile or DCB) by a stepwise increase in the concentration of the inhibitor in each subculture. Non-tolerant suspensions (I ₅₀  = 0.3 µ M ) gave rise to single cells or small clusters while tolerant cell suspensions (I ₅₀  = 30 µ M ) grown in DCB formed large clusters. The cells in these clusters were surrounded by a thick and irregular cell wall with a lamellate structure and lacking a differentiated middle lamella. Analysis of habituated cell walls by Fourier transform infrared spectroscopy and cell wall fractionation revealed: (1) a reduced amount of cellulose and hemicelluloses, mainly xyloglucan (2) qualitative and quantitative differences in pectin levels, and (3) a non-crystalline and soluble β-1,4-glucan. When tolerant cells were returned to medium lacking DCB, the size of the cell clusters was reduced; the middle lamella was only partly formed, and the composition of the cell wall gradually reverted to that obtained with non-tolerant cells. However, dehabituated cells (I ₅₀  = 12 µ M ) were 40-fold more tolerant to DCB than non-tolerant cells and were only 2.5-fold more sensitive than tolerant cells.     

Cell cycle analysis of asexual stages of erythrocytic malaria parasites

Abstract. Intra-erythrocytic Plasmodium species can be stained with the DNA binding dye, Hoechst 33342, and the distribution of DNA content determined for parasite populations by flow cytometric measurement of fluorescence. Analysis of this distribution will determine the parasitaemia (percentage of erythrocytes infected), and the percentages of trophozoite infected red blood cells, polyparasitized (trophozoite) red blood cells, and schizont/segmenter infected red blood cells. This analysis is based on the hypothesis that the asexual parasites cycle with single G₁ period, and effectively, a single S phase with no significant G₂/M period except at schizogony when the genome DNA content is equivalent to 8 N or higher, dependent on the species. Data are presented to support this model.