Technique for isolating plasmids from exopolysaccharide producing Lactobacillus spp.

Summary A large scale plasmid isolation technique is described for the isolation of plasmids from exopolysaccharide producing strains of Lactobacillus spp. Plasmids of 1.9 to 56 kb were isolated which were pure enough to be used for restriction analysis and cloning experiments.     

Function of osteocytes in bone

Although the structural design of cellular bone (i.e., bone containing osteocytes that are regularly spaced throughout the bone matrix) dates back to the first occurrence of bone as a tissue in evolution, and although osteocytes represent the most abundant cell type of bone, we know as yet little about the role of the osteocyte in bone metabolism. Osteocytes descend from osteoblasts. They are formed by the incorporation of osteoblasts into the bone matrix. Osteocytes remain in contact with each other and with cells on the bone surface via gap junction–coupled cell processes passing through the matrix via small channels, the canaliculi, that connect the cell body–containing lacunae with each other and with the outside world. During differentiation from osteoblast to mature osteocyte the cells lose a large part of their cell organelles. Their cell processes are packed with microfilaments. In this review we discuss the various theories on osteocyte function that have taken in consideration these special features of osteocytes. These are (1) osteocytes are actively involved in bone turnover; (2) the osteocyte network is through its large cell-matrix contact surface involved in ion exchange; and (3) osteocytes are the mechanosensory cells of bone and play a pivotal role in functional adaptation of bone. In our opinion, especially the last theory offers an exciting concept for which some biomechanical, biochemical, and cell biological evidence is already available and which fully warrants further investigations. © 1994 Wiley-Liss, Inc.     

Induction of apoptosis in oxygen-deprived cultures of hybridoma cells

It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures. Apoptotic or programmed cell death occurs spontaneously in late exponential phase of batch cultures. Until lately, no specific triggering factors had been identified. Recently, we observed that glutamine, cystine or glucose deprivation induced apoptosis in both hybridoma and myeloma cell lines whereas accumulation of toxic metabolites induced necrotic cell death in these cells. Other triggering factors such as oxygen deprivation might also be responsible for induction of apoptosis. In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone. The mode of cell death was studied by morphological examination of acridine orange-ethidium bromide stained cells in a 1.5 L bioreactor culture grown under anoxic conditions for 75 hours. Under such conditions, viable cell density levelled off rapidly and remained constant for 25 hours. After 45 hours of anoxia, cell viability had decreased to 30% and the dead cell population was found to be 90% apoptotic. In terms of cellular metabolism, anoxia resulted in an increase in the utilization rates of glucose and arginine, and in a decrease in the utilization rate of glutamine. The lactate production rate and the yield of lactate on glucose increased significantly while the MAb production rate decreased. These results demonstrate that glycolysis becomes the main source of energy under anoxic conditions.Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rates when reincubated in oxic conditions whereas cells incubated for 22 hours or more displayed reduced growth rates. Nonetheless, even after 22 h or 29 h of anoxia, cells reincubated in oxic conditions showed no further progression into apoptosis. Therefore, upon removal of the triggering signal, induction of apoptosis ceased.Abbreviations VNA Viable non-apoptotic cells - VA Viable apoptotic cells - NVNA Nonviable non-apoptotic or necrotic cells - NVA Nonviable apoptotic cells - CF Chromatin-free cells (late nonviable apoptotic cells) - AO Acridine orange - EB Ethidium Bromide - MAb Monoclocnal antibody - D.O. Dissolved oxygen - qMAb Specific MAb production rate (mg. (10⁹ cells)^–1.day^–1) - Specific growth rate (h^–1) - X_v Viable cell number (10⁵ cells.mL^–1) - X_t Total cell number (10⁵ cells.mL^–1) - Y_lac/glc Yield coefficient of lactate on glucose (mM lactate produced/mM glucose consumed)     

Rate of differentiation and emergence of nodal maize roots

The timing of root production is one of the parameters required for modelling the root system architecture. The objectives of this study are (1) to describe the rate of appearance of adventitious root primordia of maize and their rate of emergence out of the stem; (2) to test equations for the prediction of the rank of the phytomer on which root emergence occurs, in a wide range of field situations.Maize, cultivar Dea, was grown in controlled conditions and in the field in 1987, 1988, 1989 and 1991. Plants were regularly sampled and the following data were recorded: foliar stage, number of root primordia and number of emerged roots per phytomer. Root primordia were counted in transverse thin sections in the stem.At a single plant level, root primordia differentiation occurred sequentially on the successive phytomers, with no overlapping between two phytomers. The same was true for root emergence. Roots belonging to the same phytomer emerged at approximately the same time.At a plant population level, there was a linear relationship between the rank of the phytomer on which root primordia were differentiated and cumulated degree-days after sowing. A linear relationship was also observed between the rank of the phytomer on which roots were emerging and cumulated degree-days or foliar stage. In the range of field situations tested (several years, sowing dates and planting densities), both equations gave an accurate prediction of the timing of root emergence during the plant cycle.     

Cell cycle reentry of mammalian fibroblasts is accompanied by the sustained activation of P44mapk and P42mapk isoforms in the G1 phase and their inactivation at the G1/s transition

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that are rapidly activated in response to mitogenic stimuli. Here we examined the enzymatic activity and phosphorylation state of the individual p44^mapk and p42^mapk isoforms during early G1 and late G1 phase of the mammalian cell cycle. Release of fibroblast cells from early G1 block was accompanied by a rapid rise in the myelin basic protein (MBP) kinase activity of p44^mapk and p42^mapk, which declined slowly over several hours to reach negligible values as cells enter S phase. When cells were released from late G1 block, the activity of p44^mapk and p42^mapk increased transiently, and then rapidly declined to baseline values during G1 to S phase transition. Cells released at the G1/S boundary in a medium lacking growth factors entered S phase in the complete absence of MAP kinase activity. Unlike MAP kinases, the histone H1 kinase activity of p33^cdk2 was elevated in late G1-arrested cells and continued to increase during S phase entry. The enzymatic activation of p44^mapk and p42^mapk in both early G1 and late G1 phase was accompanied by an increase in the phosphothreonine and phosphotyrosine content of the proteins. These findings suggest that the sustained activation of MAP kinases during G1 progression and their inactivation at the G1/S transition are two regulatory processes involved in the mitogenic response to growth factors. © 1995 Wiley-Liss, Inc.     

Bone density and risk of hip fracture in men and women: cross sectional analysis.

OBJECTIVE: To determine the relative contribution of decline in bone density to the increase in risk of hip fracture with age in men and women. DESIGN: Incidence data of hip fracture from the general population were combined with the bone density distribution in a sample from the same population and with a risk estimate of low bone density known from literature. SETTING: The Netherlands. SUBJECTS: All people with a hospital admission for a hip fracture in 1993, and bone density measured in a sample of 581.4 men and women aged 55 years and over in a district of Rotterdam. MAIN OUTCOME MEASURE: One year cumulative risk of hip fracture by age, sex, and bone density measured at the femoral neck. RESULTS: A quarter of all hip fractures occurred in men. Men reached the same incidence as women at five years older. Controlled for age, the risk of hip fracture by bone density was similar in men and women. The risk of hip fracture increased 13-fold from age 60 to 80; decrease in bone density associated with age contributed 1.9 (95% confidence interval 1.5 to 2.4) in women and 1.6 (1.3 to 1.8) in men. CONCLUSIONS: The risk of hip fracture by age and bone density is similar in men and women. The decrease in bone density associated with age makes a limited contribution to the exponential increase of the risk of hip fracture with age.     

Peptide modification by introduction ofα-trifluoromethyl substituted amino acids

Summary Methodology for the synthesis and incorporation of-trifluoromethyl substituted amino acids into N- and C-terminal position of peptides is described. The incorporation of-trifluoromethyl substituted amino acids into strategical positions of peptides enhances proteolytic stability and lipophilicity. Furthermore, it improves transport rates in vivo and permeability through certain body barriers.