Regulation of Lipid Droplet Size in Mammary Epithelial Cells by Remodeling of Membrane Lipid Composition—A Potential Mechanism

Milk fat globule size is determined by the size of its precursors—intracellular lipid droplets—and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control) or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P < 0.0001). When cultured with oleic acid, 22% of the cells contained large lipid droplets (>3 μm) and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001). In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001). In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 μm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content.     

The response of stomata to CO2 relates to its effect on respiration and ATP level

External ATP enhanced stomatal opening of Commelina communis L. differently from EDTA. ATP was more effective in opening stomata than EDTA, when both were applied in amounts yielding equivalent free Ca²⁺ concentration. The stimulation by ATP depended upon its de-phosphorylation and was not due to the P₁ released. Hence an energetical contribution of external ATP appears possible. Increase in CO₂ concentration increased the stimulation of stomatal opening by ATP and diminished the internal ATP level, ATP/(ADP+AMP) ratio and respiration rate.     

Changes in the integrity of large unilamellar vesicles due to their interaction with tobacco cell suspensions

Negatively charged large unilamellar vesicles (LUV) were incubated with tobacco (Nicotiana tabacum var. xanthi) cell suspensions and with the cell-free medium of the cell suspensions. The extent of cell-LUV interaction was determined by the leakage of the LUV contents. Cells enhanced the leakage of LUV contents and this effect increased with cell age. Addition of polylysine to the reaction mixture increased even further the leakage of the LUV contents. The cell-free medium of the cell suspension also affected the integrity of the LUV. Cell-free medium, by itself, promoted leakage of LUV contents and caused a reduction in the leakage exerted by polylysine. Centrifugation (8000g) of the cell-free medium decreased its effect, heat treatment (122°C) did not alter its effect and sonication enhanced it. The effects of the cell-free medium are attributed to the presence of cell wall debris of disintegrated cells.     

Three unique base pair changes in a family with Gaucher disease

Summary Single-stranded cDNA was prepared from RNA obtained from a patient with type 1 Gaucher disease. The cDNA was amplified in vitro and analyzed by sequencing. Three base-pair changes were identified which included a G to C transversion at nucleotide 3119 of the active gene (Asp¹⁴⁰His), an A to C transversion at nucleotide 3170 (Lys¹⁵⁷Gln) and a G to A change at nucleotide 5309 (Glu³²⁶Lys). To study the mode of inheritance of the three different base-pair changes, genomic DNA was prepared from blood or skin fibroblasts of several family members. Genomic glucocerebrosidase DNA sequences were amplified and subjected to hybridization with allele-specific oligonucleotides (ASOs). The hybridization profiles demonstrated that two of the basepair changes originated from the mother and were transmitted to her two affected sons and to a grandchild, while the third base-pair change, originating from the father, was transmitted to his two affected sons, a carrier daughter and a second grandchild. Tests of other patients with Gaucher disease failed to disclose the presence of the three base-changes. This is a unique family with three base-pair changes tightly linked to Gaucher disease.     

Role of roots in regulating the growth rate and cytokinin content in leaves

The role of roots in the enhancement of cytokinin content and leaf growth of Phaseolus vulgaris plants after decapitation and partial defoliation was investigated. Partial excision of the roots of plants which were decapitated above the primary leaf node resulted in a reduction of leaf growth and soluble proteins accumulation in the primary leaves. Roots excision was done at time of decapitation and repeated 8 days later. Endogenous cytokinins, known to be involved in enhancing shoot growth, accumulated in the leaves and stems of decapitated and partially defoliated plants. Lower levels of cytokinins were detected in the leaves of decapitated plants with only a partial root system. The level of cytokinins in the roots of decapitated plants was reduced by partial root excision. The growth and accumulation of cytokinins in leaves were, however, not totally suppressed by removing a large proportion of the roots. At the commencement of the experiment the stem had a higher cytokinin content than both the leaves and roots. This suggests that the stem could be an alternative source of cytokinins to the leaves. The cytokinin complement in the leaves of decapitated plants is not identical to that in the roots. It appears that cytokinins supplied by the roots are metabolized in the leaves, or that alternatively certain cytokinins are synthesized in the leaves themselves.     

The Agrobacterium tumefaciens virC1 gene product binds to overdrive, a T-DNA transfer enhancer.

In Agrobacterium tumefaciens, a cis-active 24-base-pair sequence adjacent to the right border of the T-DNA, called overdrive, stimulates tumor formation by increasing the level of T-DNA processing. Recent results from our laboratory have suggested that the virC operon which enhances T-DNA processing probably does so because the VirC1 protein interacts with overdrive (N. Toro, A. Datta, M. Yanofsky, and E. W. Nester, Proc. Natl. Acad. Sci. USA 85:8558-8562, 1988). We report here the purification of the VirC1 protein from cells of Escherichia coli harboring a plasmid containing the coding sequences of the virC locus of the octopine Ti plasmid. By gel mobility shift and DNase I footprinting assays, we showed that this purified virC1 gene product binds to overdrive but not to the right border of T-DNA.     

An autosomal recessive nonsyndromic-hearing-loss locus identified by DNA pooling using two inbred Bedouin kindreds.

Autosomal recessive nonsyndromic hearing loss (ARNSHL) is the most common form of severe inherited childhood deafness. We present the linkage analysis of two inbred Bedouin kindreds from Israel that are affected with ARNSHL. A rapid genomewide screen for markers linked to the disease was performed by using pooled DNA samples. This screen revealed evidence for linkage with markers D9S922 and D9S301 on chromosome 9q. Genotyping of individuals from both kindreds confirmed linkage to chromosome 9q and a maximum combined LOD score of 26.2 (recombination fraction [theta] .025) with marker D9S927. The disease locus was mapped to a 1.6-cM region of chromosome 9ql3-q2l, between markers D9S15 and D9S927. The disease segregates with a common haplotype in the two kindreds, at markers D9S927, D9S175, and D9S284 in the linked interval, supporting the hypothesis that both kindreds inherited the deafness gene from a common ancestor. Although this nonsyndromic-hearing-loss (NSHL) locus maps to the same cytogenetic interval as DFNB7, it does not overlap the currently defined DFNB7 interval and may represent (1) a novel form of NSHL in close proximity to DFNB7 or (2) a relocalization of the DFNB7 interval to a region telomeric to its reported location. This study further demonstrates that DNA pooling is an effective means of quickly identifying regions of linkage in inbred families with heterogeneous autosomal recessive disorders.     

Identification of single tiny seeds ofOrobanche using RAPD analysis

The tiny seeds of parasitic weeds of the genusOrobanche can be identified by using RAPD markers. A simple procedure for DNA extraction from single seeds, 10 μg each, followed by RAPD-PCR and using specific DNA markers, leads to species identification. Seeds of five different species could be identified using this method.     

Involvement of two differenturf-s related mitochondrial sequences in the molecular evolution of the CMS-specificS-Pcf locus in petunia

In petunia, a mitochondrial (mt) locus,S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). TheS-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF,Pcf, contains parts of theatp9 andcoxII genes and an unidentified reading frame,urf-s. The second and third ORFs contain NADH dehydrogenase subunit 3 (nad3) and ribosomal protein S12 (rps12) sequences, respectively. Thenad3 andrps12 sequences included in theS-Pcf locus are identical to the corresponding sequences on the mt genome of fertile petunia. In both CMS and fertile petunia, only a single copy ofnad3 andrps12 has been detected on the physical map of the main mt genome. The origin of theurf-s sequence and the molecular events leading to the formation of the chimericS-Pcf locus are not known. This paper presents evidence indicating that two different mt sequences, related tourf-s and found in fertile petunia lines (orf-h and Rf-1), might have been involved in the molecular evolution of theS-Pcf locus. Southern analysis of mtDNA derived from both fertile and sterile petunia plants suggests that one of theseurf-s related sequences (showing 100% homology tourf-s and termedorf-h) is located on a sublimon. An additional, low-homologyurf-s related sequence (Rf-1) is shown to be located on the main mt genome 5′ to thenad3 gene. It is, thus, suggested that the sequence of events leading to the generation of theS-Pcf locus might have involved introduction of theorf-h sequence, via homologous recombination, into the main mt genome 5′ tonad3 at the region where the Rf-1 sequence is located.