Molecular Analysis of the β-Globin Gene Cluster in the Niokholo Mandenka Population Reveals a Recent Origin of the βS Senegal Mutation

A large and ethnically well-defined Mandenka sample from eastern Senegal was analyzed for the polymorphism of the beta-globin gene cluster on chromosome 11. Five RFLP sites of the 5' region were investigated in 193 individuals revealing the presence of 10 different haplotypes. The frequency of the sickle-cell anemia causing mutation (beta(S)) in the Mandenka estimated from this sample is 11.7%. This mutation was found strictly associated with the single Senegal haplotype. Approximately 600 bp of the upstream region of the beta-globin gene were sequenced for a subset of 94 chromosomes, showing the presence of four transversions, five transitions, and a composite microsatellite polymorphism. The sequence of 22 beta(S) chromosomes was also identical to the previously defined Senegal haplotype, suggesting that this mutation is very recent. Monte Carlo simulations (allowing for a specific balancing selection model, a logistic growth of the population, and variable initial frequencies of the Senegal haplotype) were used to estimate the age of the beta(S) mutation. Resulting maximum-likelihood estimates are 45-70 generations (1,350-2,100 years) for very different demographic scenarios. Smallest confidence intervals (25-690 generations) are obtained under the hypothesis that the Mandenka population is large (N(e) >5,000) and stationary or that it has undergone a rapid demographic expansion to a current size of >5,000 reproducing individuals, which is quite likely in view of the great diversity found on beta(A) chromosomes.     

Optimization of the cell wall microenvironment allows increased production of recombinant Bacillus anthracis protective antigen from B. subtilis.

The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the trans side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is D-alanylated on the secretion and stability of rPA. The potential role of the dlt operon, responsible for D-alanylation, was investigated using a Bacillus subtilis strain encoding an inducible dlt operon. We show that, in the absence of D-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of D-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of B. subtilis for the secretion of heterologous proteins.     

Optimization and mathematical modeling of the transtubular bioreactor for the production of monoclonal antibodies from a hybridoma cell line

This report describes the use of a transtubular bioreactor to study the relative effects of diffusion versus perfusion of medium on antibody production by a hybridoma cell line. The study was performed with a high-density cell culture maintained in a serum-free, low-protein medium for 77 days. It was determined that the reactor possessed a macro-mixing pattern residence time distribution similar to a continuous stirred tank reactor (CSTR). However, due to the arrangement of the medium lines in the reactor, the flow patterns for nutrient distribution consist of largely independent medium path lengths ranging from short to long. When operated with cyclic, reversing, transtubular medium flow, some regions of the reactor (with short residence times) are more accessible to medium than others (with long residence times). From this standpoint, the reactor can be divided into three regions: a captive volume, which consists of medium primarily delivered via diffusion; a lapped volume, which provides nutrients through unilateral convection; and a swept volume, which operates through bilateral convection. The relative sizes of these three volumes were modified experimentally by changing the period over which the direction of medium flow was reversed from 15 min (larger captive volume) to 9 h (larger swept volume). The results suggest that antibody concentration increases as the size of the diffusion-limited (captive) volume is increased to a maximum at around 30 min with a sharp decrease thereafter. As reflected by changes in measured consumption of glucose and production of lactate, no significant difference in cellular metabolism occurred as the reactor was moved between these different states. These results indicate that the mode of operation of the transtubular bioreactor may influence antibody productivity under serum-free, low-protein conditions with minimal effects on cellular metabolism.     

Common Cortical Loci Are Activated during Visuospatial Interpolation and Orientation Discrimination Judgements

There is a wealth of literature on the role of short-range interactions between low-level orientation-tuned filters in the perception of discontinuous contours. However, little is known about how spatial information is integrated across more distant regions of the visual field in the absence of explicit local orientation cues, a process referred to here as visuospatial interpolation (VSI). To examine the neural correlates of VSI high field functional magnetic resonance imaging was used to study brain activity while observers either judged the alignment of three Gabor patches by a process of interpolation or discriminated the local orientation of the individual patches. Relative to a fixation baseline the two tasks activated a largely over-lapping network of regions within the occipito-temporal, occipito-parietal and frontal cortices. Activated clusters specific to the orientation task (orientation>interpolation) included the caudal intraparietal sulcus, an area whose role in orientation encoding per se has been hotly disputed. Surprisingly, there were few task-specific activations associated with visuospatial interpolation (VSI>orientation) suggesting that largely common cortical loci were activated by the two experimental tasks. These data are consistent with previous studies that suggest higher level grouping processes -putatively involved in VSI- are automatically engaged when the spatial properties of a stimulus (e.g. size, orientation or relative position) are used to make a judgement.     

Chromosome size in salmon and trout

The chromosomes of the Atlantic salmon, Salmo salar (2n=58) are, on average, larger than those of the trout, S. trutta (2n=80). If the difference in chromosome size represents a permanent change in chromosome structure as between the two species the expectation is that the size difference between salmon and trout chromosomes will be maintained in the hybrid. If, alternatively, the size difference between salmon and trout chromosomes is genotypically determined the difference will not be maintained in nuclei of hybrid genotype. Measurements of a specific chromosome, S, of the salmon complement and of another, S ¹, of the trout complement in nuclei of parent species and of the hybrid show that the difference in size is maintained in hybrid nuclei. It is concluded therefore that the size difference between salmon and trout chromosomes is due to structural change rather than to genotypic control.     

The mechanism of action of aflatoxin B1 on protein synthesis: observations on malignant viral transformed and untransformed cells in culture

(1) Aflatoxin B₁ was tested against protein and nucleic acid synthesis in a number of cell lines in culture. (2) A detailed investigation was made in CV-1 cells to determine the mechanism whereby aflatoxin B₁ inhibits protein synthesis. (3) Inhibition of protein synthesis by aflatoxin B₁ was not secondary to other changes in the cell but was due to a direct action of the toxin on the polysomes. The possible site of its interaction is discussed.     

Protection of crystal-induced polymorphonuclear leukocyte membranolysis by phosphocitrate

The protection afforded by phosphocitrate, a phosphorylated polycarboxylic acid, against crystal-induced membrane damage to polymorphonuclear leukocytes was studied in vitro. Membranolysis was assessed by nitro blue tetrazolium salt reduction, lactate dehydrogenase release, and scanning electron microscopy. Phosphocitrate protected strongly against hydroxyapatite crystal-induced damage, an action attributable to crystal surface binding of phosphocitrate rather than to the membrane. The ability of phosphocitrate to prevent hydroxyapatite crystallization, together with its membrane protective effect against preformed crystals, would suggest that the compound might have a useful future role against crystal-induced arthropathies.     

Characterisation of suppressor cells generated following cryosurgery of an HSV-2-induced fibrosarcoma

Summary Cryosurgery of a primary HSV-2-induced hamster fibrosarcoma resulted in the generation of a population of suppressor cells. These cells were detectable in the spleen 1–10 days post-cryosurgery by their ability to suppress the proliferation of immunocompetent splenic T-lymphocytes following exposure to concanavalin A (Con A). The spleens of tumour-bearing (t.b.) animals which received cryosurgery 3 days previously displayed gross splenomegaly due to the generation of large numbers of highly proliferative erythroblasts. The erythroblast cells were unlikely to be the source of suppression since time course studies have demonstrated the presence of suppressor cells before and after their appearance in the spleen. The erythroblasts therefore probably reflected a response by the host to regenerate the erythrocytes lost during surgery and their presence was independent of the appearance of suppressor cells. Characterisation of the suppressor cell has revealed it to be non-adherent and esterase negative making it unlikely to be of macrophage (MØ) lineage. This was confirmed by the ability of splenic MØs from day 3 t.b. cryosurgery-treated animals to completely restore Con A-dependent T-lymphocyte proliferation following MØ depletion. As nylonwool column-eluted cells are able to suppress Con A-dependent T-lymphocyte proliferation, it seemed unlikely that B-lymphocytes play a role in cryosurgery-induced immunosuppression. These findings suggest that cryosurgery of a t.b. animal results in the generation of a population of T-lymphocytes capable of suppressing Con A-dependent T-lymphocyte proliferation, and infers that these cells contribute to the inferior prognosis following cryosurgery as compared to excision of a metastatic tumour.