Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans

Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections. In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients. Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells. It was found that indinavir impaired cryptococcal protease activity in a time- and dose-dependent fashion. The phenomenon was similarly detectable in ATCC/laboratory strains and clinical isolates. C. neoformans growth rate was also significantly reduced upon exposure to indinavir, while fungal viability was not affected and mitochondrial toxicity not detected. Furthermore, as assessed by an in vitro infection model, indinavir significantly and consistently augmented C. neoformans susceptibility to microglial cell-mediated phagocytosis and killing. Overall, by providing the first evidence that indinavir directly affects C. neoformans, these data add new in vitro insights on the wide-spectrum efficacy of PIs, further arguing for the clinical relevance of HAART against opportunistic infections in AIDS.     

What can we learn from noncoding regions of similarity between genomes?

Background  

In addition to known protein-coding genes, large amounts of apparently non-coding sequence are conserved between the human and mouse genomes. It seems reasonable to assume that these conserved regions are more likely to contain functional elements than less-conserved portions of the genome.     

Sex determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction

The aim of this study was to identify a simple, rapid method for sex determination of in vitro produced buffalo embryos, amplifying Y-chromosome-specific repeat sequences by polymerase chain reaction (PCR). Buffalo oocytes collected from slaughtered animals were matured, fertilised and cultured in vitro for 7 days. On day 7 embryos were evaluated and divided in to six groups according to developmental stage (2, 4, 8, 16 cells, morulae and blastocyst). Each embryo was stored singly in phosphate-buffered saline at -20 degrees C until PCR. Two different methods of extraction of DNA were compared: a standard procedure (ST), using a normal extraction by phenol-chloroform, isoamyl alcohol and final precipitation in absolute ethanol and a direct procedure (DT), using a commercial kit (Qiaquik-Qiagen mini blood). A pair of bovine satellite primers and two pairs of different bovine Y-chromosome-specific primers (BRY4.a and BRY.1) were used in the PCR assay on embryos and on whole blood samples collected from male and female adult buffaloes, used as control. The trial was carried out on 359 embryos (193 for ST and 166 for DT). When DNA samples from blood were amplified, the sex determined by PCR always corresponded to the anatomical sex. Embryo sexing was not possible in two embryos in ST and one embryo in DT. Both extraction protocols recovered sufficient quantities of target DNA at all developmental stages, but the time required for the ST (24 h) limits its use in embryo sexing and supports the use of commercial extraction kits (5 h).     

Differential microbial clearance and immunoresponse of Balb/c (Nramp1 susceptible) and DBA2 (Nramp1 resistant) mice intracerebrally infected with Mycobacterium bovis BCG (BCG)

In mice, the gene encoding Nramp1 (natural resistance-associated protein 1) exists in two allelic forms, differing for a point mutation. According to Nramp1 genotype, extensive literature documents a clear-cut distinction of inbred strains in two non-overlapping groups that phenotypically express resistance (Nramp1r) and susceptibility (Nramp1s) to systemic infections. Here, we provide evidence that Nramp1r (DBA/2) and Nramp1s (Balb/c) mice differently handle intracerebral infection with Mycobacterium bovis BCG. Distinct trends of microbial clearance from the brain and also different patterns of local immune responses occur, thus arguing on the involvement of Nramp1 gene product on the accomplishment of cerebral anti-mycobacterial defenses.     

Comparison of pregnancy rates with two estrus synchronization protocols in Italian Mediterranean Buffalo cows

The aim in this study was to compare two estrus synchronization protocols in buffaloes. Animals were divided into two groups: Group A (n=111) received 100 microg GnRH on Day 0, 375 microg PGF(2alpha) on Day 7 and 100 microg GnRH on Day 9 (Ovsynch); Group B (n=117) received an intravaginal drug release device (PRID) containing 1.55 g progesterone and a capsule with 10mg estradiol benzoate for 10 days and were treated with a luteolytic dose of PGF(2alpha) and 1000 IU PMSG at the time of PRID withdrawal. Animals were inseminated twice 18 and 42 h after the second injection of GnRH (Group A) and 60 and 84 h after PGF(2alpha) and PMSG injections (Group B). Progesterone (P(4)) concentrations in milk samples collected 12 and 2 days before treatments were used to determine cyclic and non-cyclic buffaloes, and milk P(4) concentrations 10 days after Artificial insemination (AI) were used as an index of a functional corpus luteum. Cows were palpated per rectum at 40 and 90 days after AI to determine pregnancies. All previously non-cyclic animals in Group B had elevated P(4) (>120 pg/ml milk whey) on Day 10 after AI. Accordingly, a greater (P<0.01) relative percentage of animals with elevated P(4) 10 days after AI were observed in Group B (93.2%) than in Group A (81.1%). However, there was no difference in overall pregnancy rates between the two estrus synchronization protocols (Group A, 36.0%; Group B 28.2%). When only animals with elevated P(4) on Day 10 after AI were considered, pregnancy rate was higher (P<0.05) for animals in Group A (44.4%) than Group B (30.3%). The findings indicated that treatment with PRID can induce ovulation in non-cyclic buffalo cows. However, synchronization of estrus with Ovsynch resulted in a higher pregnancy rate compared with synchronization with PRID, particularly in cyclic buffalo.     

Influence of rumen protein degradability on productive and reproductive performance in buffalo cows

The present study aimed to ascertain the influence of crude protein (CP) digestibility in the rumen on the quantity and quality of milk production and reproductive performance, blood (BU) and milk (MU) urea, haematological profile and vaginal mucus urea, ammonia and potassium of buffalo cows. Lactating buffaloes (n = 84), 60 days in milk, were randomly subdivided into Group C (control, n = 42) and Group T (fed a diet supplemented with Aspergillus oryzae, n = 42). In three fistulated buffalo, the diet supplemented with Aspergillus oryzae showed a decrease (P < 0.01) in protein digestibility in the rumen (79.3 vs. 45.9%). No differences were registered in productive performance. Nine buffaloes not in oestrus during the dietary treatment (Groups T1 and C1), 30 days in milk, were used to study the haematological profile and to determine milk urea and ammonia in the vaginal mucus. The animals in Group T1 had higher ammonia values in the blood (P < 0.01) but not in the vaginal mucus than Group C1. A relationship was found between MU and BU. MU was influenced by CP intake and dry matter intake. No differences between the treatments were observed in reproductive performance and the conception rate and calving interval were 37.9% and 41.4% (90 trial-day) and 449 and 419 days respectively in Groups T and C. Reproductive performance was not influenced by high levels of BU nor by blood ammonia levels, although the latter were higher in the group fed the diet supplemented with Aspergillus oryzae.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.