Sucrose dilution of frozen mouse embryos: interaction of glycerol and sucrose concentrations

Several concentrations of glycerol for cryoprotection and several concentrations of sucrose for cryoprotectant dilution were examined with frozen, thawed and cultured mouse embryos. Four hundred and eighty late morulae to early blastocyst stage embryos were collected from 35 superovulated mice (B6D2 x Swiss Webster crosses back-crossed to Swiss Webster males) 3-1/2 days after breeding. The embryos were transferred through increasing concentrations of glycerol in modified Dulbecco(1)s phosphate buffered saline (MDPBS) to reach three final concentrations of 1.0 M, 1.4 M and 1.8 M. The embryos were loaded in 0.5-ml French straws appropriately filled with the cryoprotectant and sucrose solutions for each treatment. The straws were cooled with a standard fast-freezing program to -35 degrees C, then plunged into liquid nitrogen. After 58 days of storage at -196 degrees C the straws were thawed in a 37 degrees C water bath. Cryoprotectant dilution was accomplished with a standard step-wise procedure or in the straw with one of three concentrations of sucrose solution (0.25 M, 0.5 M, 1.0 M) in MDPBS. The embryos were then washed twice in MDPBS, twice in Whitten's media for embryo culture and then placed in microdrops of Whitten's media under paraffin oil in a water saturated 5% CO(2) in air atmosphere at 37 degrees C. Embryos were observed 24 hours later for development to the expanded blastocyst stage. The proportion of embryos developing in vitro from the three glycerol concentrations were not significantly different with standard step-wise dilution procedures for glycerol removal. After step-wise cryoprotectant removal, blastocyst expansion occurred in 49%, 44% and 52% of embryos frozen in 1.0 M, 1.4 M and 1.8 M glycerol, respectively. The 1.0 M sucrose dilution of 1.0 M glycerol showed the highest development (60.5%) in vitro but was not significantly different from any of these three step-wise diluted glycerol concentrations. The step-wise dilution of the three glycerol concentrations and dilution of the 1.0 M glycerol and 1.0 M sucrose were all superior (P < 0.01) to any other dilution procedure examined.     

Polyprenyl lipid synthesis in mammalian cells expressing human cis-prenyl transferase

The level of cis-prenyl transferase activity has been implicated in controlling the level of biosynthesis of dolichol and dolichol intermediates. In this study, we isolated a cDNA encoding a human CPT (GenBank Accession No. ), which had substantial homology to other CPT isolated from human brain, bacteria, Arabidopsis, and Saccharomyces cerevisiae. Expression of this cDNA in two different insect cell lines confirmed the functionality of the protein in an in vitro assay. Western blot analysis revealed an expressed protein of approximately 38 kDa in HEK293 cells. Overexpression of the protein in HEK293 cells resulted in an increase in the level of total prenol in vivo. Furthermore, product characterization by thin layer chromatography (TLC) confirmed that the major product was a long-chain prenol with a chain length of 95 carbons. These results suggest a regulatory relationship between CPT activity and dolichol biosynthesis, and may implicate CPT in the levels of dolichol-oligosaccharide intermediate biosynthesis.     

Controlling N-linked glycan site occupancy

N-linked glycosylation, a common co-translational modification in eukaryotic cells, involves the transfer of a lipid-linked oligosaccharide onto asparagine residues in a tripeptide sequon on a nascent protein in the lumen of the endoplasmic reticulum. The attachment of an oligosaccharide unit to the polypeptide at the site of occupancy can enhance solubility, improve folding, facilitate secretion, modulate antigenicity, and increase in vivo half-life of the glycoprotein. A number of proteins exhibit variable site occupancy. The efficiency of protein N-glycosylation is dependent on the kinetics of the individual steps in the biosynthesis of the dolichol-linked oligosaccharide and the transfer of the oligosaccharide from the lipid donor substrate to the nascent polypeptide. In this review, we will discuss the role of N-linked glycan site occupancy and give an overview of the possible limitations associated with variable site occupancy. The characterization of the dolichol pyrophosphate biosynthetic pathway and the recent identification of potential rate limiting enzymes in yeast and mammalian cells has made it possible to investigate their role in site occupancy. Genetic and biochemical characterization of oligosaccharide transferase (OST) complex in yeast and mammalian cells have demonstrated the importance of specific OST subunits in protein N-glycosylation. In addition, insights into the location and residues in and around the acceptor tripeptide sequon suggest an influence on N-glycan site occupancy. Insights from these characterizations are being used to elucidate methodologies to control N-glycosylation site heterogeneity.     

Growth hormone and glucose homeostasis

Patients with active acromegaly are insulin-resistant and glucose-intolerant, whereas children with growth hormone (GH) deficiency (GHD) are insulin-sensitive and may develop fasting hypoglycaemia. Surprisingly, however, hypopituitary adults with unsubstituted GHD tend to be insulin-resistant, which may worsen during GH substitution. During fasting, which may be considered the natural domain for the metabolic effects of GH, the induction of insulin resistance by GH is associated with enhanced lipid oxidation and protein conservation. In this particular context, insulin resistance appears to constitute a favourable metabolic adaptation. The problem is that GH substitution results in elevated circadian GH levels in non-fasting patients. The best way to address this challenge is to employ evening administration of GH and to tailor the dose. Insulin therapy may cause hypoglycaemia and GH substitution may cause hyperglycaemia. Such untoward effects should be minimized by carefully monitoring the individual patient.     

The cDNA cloning and molecular evolution of reptile and pigeon lactate dehydrogenase isozymes

The cDNAs encoding lactate dehydrogenase isozymes LDH-A (muscle) and LDH-B (heart) from alligator and turtle and LDH-A, LDH-B, and LDH-C (testis) from pigeon were cloned and sequenced. The evolutionary relationships among vertebrate LDH isozymes were analyzed. Contrary to the traditional belief that the turtle lineage branched off before the divergence between the lizard/alligator and bird lineages, the turtle lineage was found to be clustered with either the alligator lineage or the alligator-bird clade, while the lizard lineage was found to have branched off before the divergence between the alligator/turtle and bird lineages. The pigeon testicular LDH-C isozyme was evidently duplicated from LDH-B (heart), so it is not orthologous to the mammalian testicular LDH-C isozymes.      

Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.     

The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii

The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a-green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a-GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a-GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer.     

Interannual variability of seasonal succession events in a temperate lake and its relation to temperature variability

The assessment of possible implications of anthropogenic climate change requires the evaluation of results obtained with complex climate models. Here we considered the problem of assessing the impact of climate variability on successional events in a lake (Plußsee) of the temperate region between January and May. We first established a statistical link between large-scale air temperature, at about 1500 m height, and the local temperature, in order to bridge the spatial gap of information obtained from global climate models and local climate which forces processes in the lake. Secondly, the local temperatures were statistically related to biologically induced dynamic features in the lake, derived from Secchi depths readings (as integrated measures). The observed relationships were compared with results from a phyto- and zooplankton population-dynamic model run under different temperature regimes. The local temperatures approximated closely the large-scale temperature. The timing of phyto- and zooplankton maxima (clearwater phase) were negatively related to the temperature. Thus, with a temperature increase both occurred earlier. The intensity of the spring algal maximum was negatively related to its timing, whereas no clear relation between the timing and intensity of the clearwater phase (zooplankton maximum) could be obtained.     

Cervical spine motion in the sagittal plane: Kinematic and geometric parameters

The kinematic function of the cervical spine has been examined previously by means of cineradiography or a sequence of lateral X-rays, usually of a flexion-extension range of motion. Interpretation of these studies, however, presents difficulties. One of the major problems is how to extract information from the X-ray images which is not only explicit, quantitative and accurate, but which is also diagnostically useful. Another problem is that as one increases the number of steps of motion between full flexion and full extension to obtain a more detailed examination, one also increases the amount of radiation exposure and the bulk of the data.

Reported here is a technique which uses one lateral-view X-ray for each of five neck positions: full flexion, full extension, and three intermediate positions. From each set of X-rays, various parameters are derived to describe two types of data: kinematic (angles and centers of rotation), and geometric (pattern of curvature). This technique has been shown capable of identifying functional abnormalities in patients with neck pain who have no structural abnormalities detectable by X-ray. Further experience will be useful in better defining various types of functional abnormalities.