New method for large-scale growth; and concentration of the Epstein-Barr viruses.

Efficacious systems are described for the large-scale growth in tissue culture and concentration of infectious (P3HR-1) and transforming (B95-8) Epstein-Barr virus. Also recorded here are our updated procedures for growing stock cultures and protocols to harvest fluids containing biologically active virus which is infectious or transforming. Various methods of concentrating biologically active Epstein-Barr virus have been evaluated. Cellular debris can be removed efficiently and rapidly from culture harvest fluids by clarification through a JCF-Z continuous-flow rotor. Efficient and reliable virus concentration was achieved by molecular filtration with Millipore Pellicon cassettes, using flow rates to 10 liters/h to produce fivefold concentrates followed by pelletization in a fixed-angle rotor. Data from recent production lots showed an average infectivity titer for P3HR-1 virus of 10(4.5) early antigen units per ml (100-fold concentrate) and 10(5.7) transforming units per ml (200-fold concentrate) for B95-9 virus lots.     

Mutually exclusive recombination of wild-type and mutant loxP sites in vivo facilitates transposon-mediated deletions from both ends of genomic DNA in PACs

Recombination of wild-type and mutant loxP sites mediated by wild-type Cre protein was analyzed in vivo using a sensitive phage P1 transduction assay. Contrary to some earlier reports, recombination between loxP sites was found to be highly specific: a loxP site recombined in vivo only with another of identical sequence, with no crossover recombination either between a wild-type and mutant site; or between two different mutant sites tested. Mutant loxP sites of identical sequence recombined as efficiently as wild-type. The highly specific and efficient recombination of mutant loxP sites in vivo helped in developing a procedure to progressively truncate DNA from either end of large genomic inserts in P1-derived artificial chromosomes (PACs) using transposons that carry either a wild-type or mutant loxP sequence. PAC libraries of human DNA were constructed with inserts flanked by a wild-type and one of the two mutant loxP sites, and deletions from both ends generated in clones using newly constructed wild-type and mutant loxP transposons. Analysis of the results provides new insight into the very large co-integrates formed during P1 transduction of plasmids with loxP sites: a model with tri- and possibly multimeric co-integrates comprising the PAC plasmid, phage DNA, and transposon plasmid(s) as intermediates in the cell appears best to fit the data. The ability to truncate a large piece of DNA from both ends is likely to facilitate functionally mapping gene boundaries more efficiently, and make available precisely trimmed genes in their chromosomal contexts for therapeutic applications.     

The effects of the menstrual cycle, pregnancy and early lactation on haematology and plasma biochemistry in the baboon (Papio hamadryas)

The changes in the haematology and clinical biochemistry associated with the different stages of the menstrual cycle, gestation and lactation in the baboon (Papio hamadryas) were evaluated in a prospective longitudinal study. Serial EDTA and heparin blood samples were collected from 12 baboons. Haemoglobin concentration, haematorcrit, red blood cell and white blood cell counts were decreased in the luteal compared to the follicular phase (P<0.001); the reverse effect was observed for platelet count, total protein and albumin concentrations. The changes in plasma concentrations of sodium, potassium, urea, creatinine and cholesterol and plasma osmolality were characterized by reductions (P<0.01) in early pregnancy which were maintained throughout gestation. Plasma concentrations of total protein, albumin and alkaline phosphatase, as well as haemoglobin, haematocrit and red cell count were reduced (P<0.001) from mid-gestation. Platelet count and plasma calcium concentration fell continuously throughout gestation (P<0.001). Plasma triglycerides were lower and plasma iron was higher (P<0.01) in gestation compared to the phases of the menstrual cycle and lactation. By 1 week post partum, all parameters except haemaglobin had returned to pre-conception levels.     

Fetal ultrasonography: Normal biometric ranges in the baboon (Papio hamadryas)

Normal biometric ranges for fetal growth in a captive breeding baboon (Papio hamadryas) colony are described. Measurements include crown-rump length, biparietal diameter, binocular distance, head circumference, abdominal circumference, femur length and amniotic fluid index. The pattern of fetal growth is compared with other baboon subspecies and man. The uses and limitations of such data for breeding colony management and optimum utilisation of experimentally derived data are discussed.     

Cyclical changes in the renin-angiotensin-aldosterone system during the menstrual cycle of the baboon (Papio hamadryas)

Abstract: This study characterizes the renin-angiotensin-aldosterone system during the normal menstrual cycle in the baboon. Ten animals received a daily dose of an ACE inhibitor or placebo in a randomized blind cross-over design. Data were obtained during the mid-follicular and early luteal phases of normal non-pregnant menstrual cycles. All examinations and blood collections were performed with ketamine sedation: 7–kg by im injection. Blood pressure was recorded by sphygmomanometer. Serum ACE activity was measured by spectrophotometry. Aldosterone (ALDO), angiotensin I (AI), and angiotensin II (AII) were measured by radioimmunoassay. Plasma renin activity (PRA) was measured by AI generation. The renin-angiotensin-aldosterone system was found to be activated in the follicular phase and suppressed during the luteal phase of the normal non-pregnant menstrual cycle in the baboon.     

Minimal cross-recombination between wild-type and loxP511 sites in vivo facilitates truncating both ends of large DNA inserts in pBACe3.6 and related vectors

Contrary to several earlier reports, we find that cross-recombination between wild-type and the mutant loxP511 sites is <0.5% of that between two wild-type sites if Cre protein is expressed by phage P1 during an infection. The finding enabled us to develop a procedure to truncate DNA progressively from both ends of large genomic inserts flanked by these two loxP sites in pBACe3.6 and related vectors with transposons carrying either a wild-type or a loxP511 sequence. Newly constructed loxP511 transposons contained either a kanamycin resistance gene or no marker. Insert DNA ends in deletions were sequenced with primers unique to each transposon-end remaining after the respective recombination. End-sequencing 223 deletions confirmed that the low level of cross-recombination, observed between those sites during the P1 transductions, does not complicate the procedure: truncations from the unintended end of genomic inserts did not occur. Multiple BACs pooled together could also be processed in a single tube to make end-deletions. This deletion technology, utilizing the very minimal cross-recombination between the mutant and wild-type loxP sites of most BAC clones in the public domain and a heterologous one inserted as a transposon, should facilitate functionally mapping long-range gene regulatory sequences and help to isolate genes with defined functional boundaries in numerous projects including those of therapeutic interest.     

Production of Diamino propionic acid ammonia lyase by a new strain of Salmonella typhimurium PU011

Background  

Seeds of the legume plant Lathyrus sativus, which is grown in arid and semi arid tropical regions, contain Diamino Propionic acid (DAP). DAP is a neurotoxin, which, when consumed, causes a disease called Lathyrism. Lathryrism may manifest as Neurolathyrism or Osteolathyrism, in which the nervous system, and bone formation respectively, are affected. DAP ammonia lyase is produced by a few microorganisms such as Salmonella typhi, Salmonella typhimurium and Pseudomonas, and is capable of detoxifying DAP.     

Biochemistry and haematology values for the baboon (Papio hamadryas): the effects of sex, growth, development and age.

A retrospective study evaluated the influence of sex and age on plasma biochemistry and haematology parameters in a captive-bred colony of baboons. Over 1,140 ETDA and heparin blood samples were obtained from 160 clinically normal baboons between the ages of 11 months and 11 years. Data for these blood tests were analysed for the effects of sex, age and sex age interactions. Sex, age and sex age interactions were detected for many plasma biochemistry and haematological parameters. The reference range values for platelets, white-blood cells and mean corpuscular volume and plasma chloride, glucose, total protein and iron were higher (P < 0.01) and red blood cell, plasma sodium, potassium, total CO2, creatinine, urea, total bilirubin, albumin, alkaline phosphate, gamma glutamyl transpeptidase and phosphate were lower (P < 0.01) in the female compared to the male population. Sex age interactions (P < 0.05) were seen with haemoglobin, white blood cells, haematocrit, mean corpuscular volume, sodium, creatinine, urea, calcium, phosphate, total bilirubin, total protein alkaline phosphatase, the liver enzymes and triglycerides. Plasma alkaline phosphatase was highest ( > 800 micro/l) in young juveniles of both sexes; creatinine was higher in older ( > 4 years) compared to younger baboons of the same sex (P < 0.05). Plasma cholesterol and triglycerides were greater (P < 0.01) in young baboons compared to older animals.     

Modelling the transition from simple to complex Ca2+oscillations in pancreatic acinar cells

A mathematical model is proposed which systematically investigates complex calcium oscillations in pancreatic acinar cells. This model is based on calcium-induced calcium release via inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) and includes calcium modulation of inositol (1,4,5) trisphosphate (IP₃) levels through feedback regulation of degradation and production. In our model, the apical and the basal regions are separated by a region containing mitochondria, which is capable of restricting Ca²⁺ responses to the apical region. We were able to reproduce the observed oscillatory patterns, from baseline spikes to sinusoidal oscillations. The model predicts that calcium-dependent production and degradation of IP₃ is a key mechanism for complex calcium oscillations in pancreatic acinar cells. A partial bifurcation analysis is performed which explores the dynamic behaviour of the model in both apical and basal regions.     

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.