The identification of a normal rat gene located close to the gene for the potential myoepithelial cell calcium-binding protein, p9Ka

The potential calcium-binding protein p9Ka is related to S-100 protein and the vitamin D-dependent intestinal calcium-binding protein. p9Ka accumulates abundantly in cultured rat mammary myoepithelial-like cells but is very much less abundant in the parental cuboidal epithelial cells. p9Ka mRNA is found in normal rat mammary gland, and preliminary experiments suggest that it is found in the mammary myoepithelial cells. A 17-kilobase pair fragment of cloned normal rat DNA contains the gene for p9Ka, but it also contains the gene for two additional polypeptides of molecular mass 6 kDa that are resolved as two isoelectric focusing variants by two-dimensional gel electrophoresis. These two isoelectric focusing variants correspond to two abundant polypeptides present in the cultured myoepithelial cells and probably arise from postsynthetic modification of the product of a single gene. The mRNA for the product of this gene and the p9Ka mRNA are both found in the normal rat mammary gland, but these two mRNAs are differentially expressed in certain tumor-derived rat cell lines.     

Control of type IV collagen production in rat mammary epithelial and myoepithelial-like cells

A rat mammary myoepithelial-like cell line (Rama 401) produces 3.5 times more type IV collagen than a mammary epithelial cell line (Rama 25), as measured by the formation of protein hydroxyproline. However, using quantitative "dot" hybridization techniques, the level of poly (A)-containing mRNA hybridizing to a type IV collagen cDNA probe is only 50% higher in Rama 401 cells than in Rama 25 cells. The total amount of hydroxyproline synthesized per cell by the two cell lines is similar. However, in the Rama 25 cells approximately 70% of the hydroxyproline is found as free hydroxyproline against 13% for Rama 401 cells. When Rama 25 cells are grown on collagen gels, they accumulate 2.5-fold more type IV collagen. However, type IV collagen mRNA levels are only 30% higher in Rama 25 cells grown on collagen. The total amount of hydroxyproline synthesized is the same as cells grown on plastic, whereas the extent of collagen degradation is reduced from 71% to 30% in cells grown on collagen gels. No degradation of type IV collagen can be detected in the culture medium of Rama 25 cells. These results indicate that the increased accumulation of type IV collagen in Rama 401 cells is not due to increased synthesis but to a decreased rate of intracellular degradation, and that for Rama 25 cells, the extracellular matrix modulates type IV collagen production by regulating the rate of intracellular collagen degradation.     

The biosynthesis of ribulose bisphosphate carboxylase. Uncoupling of the synthesis of the large and small subunits in isolated soybean leaf cells

Isolated leaf cells from soybean (Glycine max) incorporate [35S]methionine into protein at a linear rate for at least 5h. Analysis of the products of incorporation by one-dimensional and two-dimensional polyacrylamide gel electrophoresis shows that major products are the large and small subunits of the chloroplast enzyme, ribulose bisphosphate carboxylase. The large subunit is synthesized by chloroplast ribosomes and the small subunit by cytoplasmic ribosomes. Addition of chloramphenicol to the cells reduces incorporation into the large subunit without affecting incorporation into the products of cytoplasmic ribosomes. Addition of cycloheximide or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide stops incorporation into the small subunit, but large subunit continues to be made for at least 4 h. For accurate estimates of incorporation into the large subunit, it is essential to use two-dimensional gel electrophoresis, because the large subunit region on one-dimensional gels is contaminated with the products of cytoplasmic ribosomes. Newly synthesized large subunits continue to enter complete molecules of ribulose bisphosphate carboxylase in the absence of small subunit synthesis. These results suggest that, in contrast to the situation in algal cells, the synthesis of the two subunits of ribulose bisphosphate carboxylase in the different subcellular compartments of higher plant cells is not tightly coupled over short time periods, and that a pool of small subunits exists in these cells. The results are disucssed in relation to possible mechanisms for the integration of the synthesis of the large and small subunits of ribulose bisphosphate carboxylase.     

Isolation of a Potential Neural Stem Cell Line from the Internal Capsule of an Adult Transgenic Rat Brain

A thermosensitive mutation of simian virus 40 large T antigen (LTA) gene, the tsA58 gene, was cloned downstream of the 6-kbp neurofilament light chain promoter in pPOLYIII and injected into the pronucleus of fertilised oocytes of Sprague-Dawley rats to develop a strain harbouring six copies of the transgene. Immunocytochemical staining of hemizygous adult tissues with antibodies to the C-terminus of LTA showed that the inactive form of LTA was expressed only in the fibres of the internal capsule and in the choroid plexus of the brain. Culturing the former region at 33 degrees C, the permissive temperature for LTA, yielded a cell line, NF2C, which produced active LTA and grew at 33 degrees C but which produced only inactive LTA and eventually died at the non-permissive temperature of 39 degrees C. This clonal cell line was heterogeneous at 33 degrees C, producing the precursor neuronal cell marker nestin and the glial-specific markers glial fibrillary acidic protein, vimentin and S100A1, as well as weakly producing the neuronal cell markers 68-kDa neurofilament protein (NF68) and microtubule-associated protein 2 (MAP2) in different subpopulations of cells. However, at 39 degrees C, the cells produced dendritic, neuronal-like processes and elevated levels of NF68 and MAP2, as well as the neuronal markers synaptophysin, neurone-specific enolase, and low levels of tau, all determined by western blotting and immunofluorescent staining. Basic fibroblast growth factor enhanced the growth of the cells at 33 degrees C but also enhanced the formation of dendritic neuronal-like processes at 39 degrees C. It is suggested that NF2C represents a potential stem cell line from adult brain that expresses precursor and glial cell markers at 33 degrees C but undergoes partial differentiation to a neuronal cell phenotype at 39 degrees C.     

Utilization of milk energy by suckling mink kits

A total of 36 mink dams and their litters of 3, 6 or 9 kits were used for determination of milk intake of the suckling young by means of deuterium dilution technique, and chemical composition of milk and of kit bodies. Measurements were performed during lactation weeks 1?–?4, each week with 3 dams with each litter size. Milk intake was determined over a 48?h measurement period, and by the end of this milk samples were collected and 2 kits (litters of 6 and 9) or 1 kit per litter (litters of 3) were killed for body chemical composition. Based on the results, different models were applied for calculation of the energetic efficiency of milk. Dam milk yield increased steadily from week 1 until week 3 but only slightly from week 3 to 4. The increase declined with increasing litter size, and for dams suckling 9 kits the increment from week 3 to week 4 was only 2?g. The dry matter content of milk increased significantly as lactation progressed, being reflected in crude protein increasing from 6.9% in lactation week 1 to 8.1% in week 4. Milk fat increased concomitantly from 5.6% to 8.0%. In kit bodies, crude protein content increased from 9.4% in week 1 to about 12% in weeks 3 and 4. Body fat content increased from week 1 (4.1%) to week 3 (8.4%) and then declined in week 4 (7.1%). Animals suckled in litters of 3 kits had the highest milk intake and live weight and kits suckled in litters of 9 had the lowest milk intake, live weight and daily gain. In terms of milk intake per g gain kits in litters of 6 were the most efficient, with 4.1?g milk per g body gain. The metabolizable energy requirement for maintenance (MEm) was estimated to 448 kJ/kg^0.75 and the efficiency of utilization of ME for body gain (k_g) to 0.67, the estimates being higher (MEm) or in good agreement with previous findings (k_g) in suckling mink kits.     

Relationship between photochemical reflectance index and leaf ecophysiological and biochemical parameters under two different water statuses: towards a rapid and efficient correction method using real‐time measurements

The use of the photochemical reflectance index (PRI) as a promising proxy of light use efficiency (LUE) has been extensively studied, and some issues have been identified, notably the sensitivity of PRI to leaf pigment composition and the variability in PRI response to LUE because of stress. In this study, we introduce a method that enables us to track the short‐term PRI response to LUE changes because of photosynthetically active radiation (PAR) changes. The analysis of these short‐term relationships between PRI and LUE throughout the growing season in two species (Quercus robur L. and Fagus sylvatica L.) under two different soil water statuses showed a clear change in PRI response to LUE, which is related to leaf pigment content. The use of an estimated or approximated PRI0, defined as the PRI of perfectly dark‐adapted leaves, allowed us to separate the PRI variability due to leaf pigment content changes and the physiologically related PRI variability over both daily (PAR‐related) and seasonal (soil water content‐related) scales. The corrected PRI obtained by subtracting PRI0 from the PRI measurements showed a good correlation with the LUE over both of the species, soil water statuses and over the entire growing season.     

Independently evolving species in asexual bdelloid rotifers

Asexuals are an important test case for theories of why species exist. If asexual clades displayed the same pattern of discrete variation as sexual clades, this would challenge the traditional view that sex is necessary for diversification into species. However, critical evidence has been lacking: all putative examples have involved organisms with recent or ongoing histories of recombination and have relied on visual interpretation of patterns of genetic and phenotypic variation rather than on formal tests of alternative evolutionary scenarios. Here we show that a classic asexual clade, the bdelloid rotifers, has diversified into distinct evolutionary species. Intensive sampling of the genus Rotaria reveals the presence of well-separated genetic clusters indicative of independent evolution. Moreover, combined genetic and morphological analyses reveal divergent selection in feeding morphology, indicative of niche divergence. Some of the morphologically coherent groups experiencing divergent selection contain several genetic clusters, in common with findings of cryptic species in sexual organisms. Our results show that the main causes of speciation in sexual organisms, population isolation and divergent selection, have the same qualitative effects in an asexual clade. The study also demonstrates how combined molecular and morphological analyses can shed new light on the evolutionary nature of species.     

How do roots penetrate strong soil?

The mechanical and physiological bases for root growth against high mechanical impedance are reviewed. The best estimates of maximum axial root growth pressure (_max) in completely impeded pea roots appear to be from 0.5 to 0.6 MPa, which results from a turgor pressure of about 0.8 MPa. When roots are incompletely impeded, a range of responses has been reported. Roots do not change elongation rate in a simple mechanical way in response to changes in mechanical impedance. Instead, ethylene might play a key role in mediating an increase in root diameter and a decrease in elongation rate. These changes persist for some hours or days after impedance is removed. Differences between species in their ability to penetrate strong soil layers are not related to differences in _max, but appear to be due to differences in root diameter. In rice, differences between cultivars in the ability of their roots to penetrate strong wax layers are not related to their elongation rates through uniformly strong media. Differences between species or cultivars in their ability to penetrate strong layers may be due to differences in the tendency of roots to deflect or buckle when they grow from a weak to a strong environment.