Electrophoretic and chromatographic evidence for allelic polymorphisms in the river buffalo α-globin gene complex

Isoelectric focusing in the ultranarrow immobilized (7.1–7.5) pH gradient (IPG) of hemoglobin and high-performances liquid chromatography (HPLC) of globin chains were used to investigate Hb polymorphism in Italian river buffalo. Six different phenotypes, each characterized by two or four different Hbs, were detected by IPG, whereas two different^IIα-globin chains were separated from two different^Iα-chains by HPLC. Two α-chains (^Iα¹ and^IIα³), and Hbs with similar mobilities (Hb1 andHb3), were associated with the AA Hb phenotype: two α-chains (^Iα² and^IIα⁴), and Hbs with different mobilities (Hb2 andHb4), were associated with the BB phenotype: two sets of doublet Hbs were associated with the AB phenotype, thus suggesting allelic polymorphisms at the two α loci. An allele at the β locus is responsible for increasing to as many as eight the number of different Hbs, thus further complicating the notable Hb polymorphism of the river buffalo.     

Eland, buffalo, and wild pigs: were Middle Stone Age humans ineffective hunters?

Patterns of faunal exploitation play a central role in debates concerning the behavioral modernity of Middle Stone Age (MSA) peoples. MSA foragers have been portrayed as less effective hunters than their Later Stone Age (LSA) successors on the basis of relative species abundances from ungulate assemblages in southern Africa. Specifically, MSA hunters are said to focus on docile eland while avoiding more aggressive prey, particularly buffalo and wild pigs. To evaluate these arguments and compare subsistence behavior, I present a quantitative examination of 51 MSA and 98 LSA ungulate assemblages from southern Africa to show that: (1) with respect to ungulate exploitation, MSA diet breadth may have exceeded LSA diet breadth, (2) ungulate assemblage evenness is equivalent in the MSA and LSA, (3) eland, buffalo, and wild pig are equally abundant in the MSA and LSA, and (4) large ungulate prey are more common in the MSA than in the LSA. With few exceptions, the broad patterns, which sample a range of geographic and environmental contexts, are supported by an environmentally controlled comparison of Middle and Later Stone Age faunas that accumulated under interglacial conditions along the southern African coastline. When interpreted within a foraging theory framework, these differences suggest that MSA hunters enjoyed increased meat yields due to elevated encounter rates with large prey. These results need not imply cognitive differences, but are consistent with an increase in human populations from the Middle to Later Stone Age, which resulted in diminished abundances of large ungulates.     

Acetoxy drug: protein transacetylase of buffalo liver—characterization and mass spectrometry of the acetylated protein product

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3–3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K_m (1667 μM) and V_max (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3–3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3–3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3–3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3–3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys₅₁, Lys₈₂, Lys₁₂₃, Lsy₁₈₁, Lys₁₉₁ and Lys₂₁₀) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.     

Characterization of β-casein gene in Indian riverine buffalo

The study aimed at characterization of buffalo β-casein gene and its promoter by PCR-SSCP analysis. Complete β-casein exon VII region analysis revealed two SSCP band patterns, with pattern-I representing predominant allele B (85%) present in homozygous (genotype BB) condition and pattern-II representing a rare allele A1 present in heterozygous condition (genotype A1B). Sequencing of two patterns revealed three nucleotide substitutions at codon 68, 151 and 193 of exon VII. The cDNA sequence of buffalo β-casein gene indicated three further nucleotide substitutions between allele A1 and B at codon 10, 39, and 41. Analysis of β-casein proximal promoter region (− 350 upstream to + 32) revealed four SSCP band patterns. These SSCP patterns corresponded to nucleotide substitutions at seven locations within 382 bp 5′ UTR region of β-casein gene. Haplotype analysis suggested pattern-I of exon VII (wild type) was associated with three types of promoters and pattern-II of exon VII (rare type) corresponded to one exclusive type of promoter. The study suggested two haplotypes of exon VII and four haplotypes of promoter for buffalo β-casein.     

Detection of estrous behavior in buffalo heifers by radiotelemetry following PGF2α administration during the early or late luteal phase

This study examined the usefulness of radiotelemetry for estrous detection in buffalo heifers and the impact of prostaglandin F_2α (PGF_2α) administration during the early or late luteal phase on estrous behavior and ovulatory follicle variables. Induction of estrus with PGF_2α at a random stage of the estrous cycle was followed by the arbitrary division of heifers into groups receiving a second dose of PGF_2α during either the early (n = 33) or late (n = 17) luteal phase (6–9 or 11–14 days after estrus, respectively) for the induction of synchronized estrus. The electronic detection of synchronized estrus by radiotelemetry was confirmed using ultrasonography every 6 h until ovulation. Radiotelemetry was 90% efficient and 100% accurate for estrous detection. Intervals between the PGF_2α dose and the beginning of synchronized estrus (40.7 ± 10.9 vs. 56.7 ± 12.8 h) or ovulation (70.0 ± 11.3 vs. 85.6 ± 12.5 h) were shorter (P < 0.05) for heifers receiving PGF_2α during the early luteal phase. PGF_2α administration during the early or late luteal phase produced similar (P > 0.05) results for the duration of estrus, the intervals from the beginning or end of estrus to ovulation, the number and duration of mounts per estrus, the duration of mounts, the diameter of the ovulatory follicle and the luteal profile on day 5 after estrus. In conclusion, radiotelemetry was a suitable tool for the efficient and accurate detection of estrus in buffalo heifers. Treatment with PGF_2α during the early luteal phase had a shorter interval to synchronized estrus and ovulation; however, estrous behavior, ovulatory follicle dynamics and subsequent luteal activity were similar following PGF_2α administration during the early or late luteal phase.     

Genetic studies of water buffalo blood markers. I. Red cell acid phosphatase,albumin, catalase,red cell α-esterase-3, group-specific component,and protease inhibitor

We have developed the methodologies for typing and family studies to establish the modes of inheritance of water buffalo red cell acid phosphatase (Acp), protease inhibitor (Pi), and group-specific component (Gc) on isoelectric focusing and albumin (Alb), red cell -esterase-3 (Est-3), and catalase (Cat) on polyacrylamide gel electrophoresis. Family studies showed that Pi, Gc, Alb, and Cat are coded by autosomal genes with two codominant alleles, while Est-3 is autosomal with two codominant alleles and a recessive null allele and Acp exhibits three codominant alleles.This project was funded by the Australian Centre for International Agricultural Research through Grant PN 8364 and the Malaysian programme for Intensification of Research in Priority Areas through Grant IRPA 1-07-05-057.     

α-Tocopherol and l-ascorbic acid increase the in vitro development of IVM/IVF swamp buffalo ( Bubalus bubalis) embryos

This study was conducted to investigate the effects of capacitating agents added at in vitro fertilization (IVF) and antioxidants supplemented during in vitro culture (IVC) on the development of buffalo embryos. In experiment I, in vitro embryo development of buffalo embryos was compared when the IVF medium was supplemented with heparin, caffeine and calcium ionophore A23187 either alone or in combination. There was no significant difference (P > 0.05) in the cleavage rates of oocytes among the treatment groups but the development rate to the blastocyst stage and the cell numbers of blastocyst in the heparin-treated group were significantly higher (P < 0.05) than that of other treatments. In experiment II, in vitro embryo development of buffalo embryos was compared when IVC medium was supplemented with either α-tocopherol (250 and 500 μM) or l-ascorbic acid (250 and 500 μM). The rate of development to the blastocyst stage of embryos cultured in medium supplemented with 250 μM α-tocopherol (33%, 41/123) and 250 μM l-ascorbic acid (31%, 38/123) was significantly higher (P < 0.05) than that of those cultured in medium alone (19%, 20/108) but not significantly different (P < 0.05) from medium supplemented with either 500 μM α-tocopherol (24%, 30/123) or 500 μM l-ascorbic acid (25%, 33/133). These results suggest that buffalo spermatozoa treated with heparin were suitable for IVF and that α-tocopherol and l-ascorbic acid added during IVC increased the rate of buffalo embryo development.     

Lactate dehydrogenase β processed pseudogene in river buffalo and other mammalian species

In the present study a primer pair originally designed to amplify a DNA segment of the lactate dehydrogenase β (LDHβ) parent gene was tested in river buffalo. The primer pair amplified a 318 bp DNA segment. The DNA sequence of this segment was determined and compared with the mammalian whole genome sequences in Genbank database for human, cattle and mouse. Blast data analysis showed that the sequence of the buffalo amplified DNA segment aligns with LDHβ parent genes of cattle, mouse and human at four scattered sites representing the last 23 bases of exon 2, exon 3, exon 4 and the first three bases of exon 5. Results also revealed that the sequence of buffalo DNA segment is 98%, 88% and 85% similar to a DNA segment of LDHβ processed pseudogene (LDHβP) of cattle, mouse and humans, respectively. These findings indicate that the amplified DNA segment does not belong to LDHβ parent gene and that as in human, cattle and mouse, the river buffalo has an LDHβ pseudogene of the processed type.     

Phytochemicals: the good, the bad and the ugly?

Phytochemicals are constitutive metabolites that enable plants to overcome temporary or continuous threats integral to their environment, while also controlling essential functions of growth and reproduction. All of these roles are generally advantageous to the producing organisms but the inherent biological activity of such constituents often causes dramatic adverse consequences in other organisms that may be exposed to them. Nevertheless, such effects may be the essential indicator of desirable properties, such as therapeutic potential, especially when the mechanism of bioactivity can be delineated. Careful observation of cause and effect, followed by a coordinated approach to identify the responsible entities, has proved extremely fruitful in discovering roles for phytochemical constituents. The process is illustrated by selected examples of plants poisonous to animals and include the steroidal alkaloid toxin of Veratrum californicum (Western false hellebore), piperidine alkaloids of Lupinus species (lupines), and polyhydroxy indolizidine, pyrrolizidine and nortropane alkaloids of Astragalus and Oxytropis species (locoweeds), Castanospermum australe (Moreton Bay chestnut) and Ipomoea species (morning glories).     

Jiuzhaigou, the Picturesque Valley

Jiuzhaigou covers an area of 720 square kilometers, with an outer-ring protect zone of 600 square kilometers, among which a great majority part is covered with forest. The main gully, more than 50 kilometers long, consists of Shuzheng, Rize arid Zechawa three gullies, forming a shape of "Y". At the end of Zechawa gully lies Changhai (the lake is called "haizi" by the local people) with an eleva-     

C-terminal region of human p53 attenuates buffalo p53 N-terminal-specific transactivation of p21 promoter by modulating tetramerization of the protein

Here, we have studied in p53 null H1299 lung carcinoma cells, the dominant-negative effect of human p53 (h-p53) on buffalo p53 (b-p53) induced nuclear transactivation-dependent function. Recently, we have isolated and cloned the full-length cDNA of buffalo p53. Buffalo and human p53 proteins exhibit a high degree of structural and functional similarities. In transiently transfected H1299 cell line b-p53 appeared to be more sensitive to Mdm2-mediated degradation as compared to h-p53, although its ability to transactivate p21 promoter was stronger than that of the human counterpart. This higher transactivation ability of b-p53 was lost in the presence of h-p53 suggesting, a dominant-negative effect of h-p53 on b-p53’s transactivation of p21 promoter. Both human and buffalo p53 proteins could hetero-oligomerize but the b-p53 could tetramerize much faster than the h-p53. A chimeric cDNA construct of human p53 was made where the 1–260 bp N-terminus was replaced with buffalo p53 counterpart and expressed in H1299 cell line. The tetramerization ability of the chimeric p53 protein was comparable to that of h-p53. Properties of b-p53 like stronger p21 transactivation and super sensitivity to Mdm2 mediated degradation were lacking in the chimeric protein. Thus, it is suggested that faster ability of tetramerization as well as higher transactivation property of buffalo p53 is determined by the interplay of N- and C-terminal domains through macromolecular interactions.     

Tau phosphorylation, tangles, and neurodegeneration: the chicken or the egg?

Pathological aggregation of the microtubule-associated protein tau is a common feature of many neurodegenerative diseases. Although tau aggregation is associated with abnormal tau phosphorylation, the role of phosphorylation in the initiation of neurodegeneration has been unclear. Now, several animal models and data from human patients provide converging evidence that aberrant tau phosphorylation can cause a neurodegenerative phenotype similar to that seen in human neurodegenerative diseases.     

APPENDIX： Statement delivered from the floor at the Extraordinary Session of the International BioethicsCommittee, UNESCO, Paris, 27- 29 April, 2004

I have heard, over the last two days, from the learned and respected scientists and philosophers here that we must not, indeed cannot expand bioethics to apply to the biosphere, i. e. bioethics is not bio at all, it is anthropocentric ethics.     

Vegetation changes during the Holocene in the North Ibersá, Corrientes, Argentina

Vegetation changes during the Holocene in the North Iberá, Corrientes, Argentina. Wetlands are very important sites for palynological studies, since they represent one of the most suitable environments for fossil pollen preservation. The aim of this work was to determine, by palynological analysis of lacustrine sediments, the vegetal communities and the predominant environment during the Holocene in NW of Iberá. Two lagoons were studied: San Sebastián and San Juan Poriahú. Sediment samples were obtained with witness using a "Levingstone square-rod sampler", processed with Faegri e Iversen techniques and dated with C14. The palynological graphs were divided in zones using the Tilia program. The palynological analysis allowed visualizing diverse changes in the vegetation: from 6 140 +/- 50 to 5 170 +/- 100 a. C., the NW of Iberá was characterized by marsh-herbaceous vegetation and arboreal vegetation typical of dry vegetation. From 5 170 +/- 100 to 3 460 +/- 60 a. C., a decrease in the species frequency, typical of wet environments, is produced, and the clogging of the waterbody, from 3460 +/- 60 a. C. onwards, while continuing the dominance of herbaceous vegetation typical of these environments, the arboreal pollen, indicates the beginning of a hygrophilous forest development.