Determination of size distribution of elliptical microvessels from size distribution measurement of their section profiles

In transmission electron microscopy, microvessels (MVs) are studied as profiles on ultrathin sections. To determine MV sizes from measurements made on MV profiles, an assumption must be made about MV shape, a circular cylinder being used to approximate the latter on limited lengths. However, this model is irrelevant in case MVs have some flatness. The elliptical cylinder model is preferable, although relationships between the cylinder profile (two-dimensional; 2D) and its true (three-dimensional; 3D) sizes are not yet known. We have obtained the 2D/3D functions that express the relationships between such profile sizes as the minor radius (Y), major radius (X), axial ratio (X/Y), area (S), and perimeter (P) on the one hand, and the corresponding MV sizes (Y(0), X(0), X(0)/Y(0), S(0), and P(0)) on the other. The 2D/3D functions make it possible to derive elliptical MV sizes from section profile size distributions, probability density functions (PDFs) for the latter being determined. We have applied the 2D/3D functions in studying axial ratios of thyroid hemocapillaries. A factual X/Y frequency histogram has been constructed and fitted by theoretical X/Y PDFs plotted for different sets of capillary sizes. The thyroid capillaries have been revealed to be clustered, 72.7% of them having X(0)/Y(0) approximately 1.6, 17.6%, X(0)/Y(0) approximately 1.0, and 9.7%, X(0)/Y(0) approximately 3.2. The proposed technique is instrumental in precise modeling of microcirculatory network geometry.     

Probability density functions for axial ratios of sectioning profiles of anisotropically arranged elliptical microvessels

The article theoretically regards probability density functions (PDFs) for axial ratio (X/Y) of sectioning profiles of elliptical microvessels (MVs) arranged with anisotropy in a biological tissue volume. A technique for the PDF_X/Y calculations in anisotropy of the elliptical MVs is described. The essence of this technique is introducing anisotropy in PDF(α,φ), i.e. the function of the joint distribution of the polar and planar angles α and φ, which define mutual orientation of the elliptical MVs and sectioning planes. With the aid of this technique, the anisotropy cases are studied with PDF(α,φ) given by pair combinations of the following distributions: (i) a uniform distribution of the angles α and/or φ, (ii) the angle α distribution with , and (iii) Gaussian distributions of the α or φ values. Specifically, PDF_X/Y curves are obtained for MVs with the true, or three-dimensional, axial ratio X₀/Y₀=2.0, and the anisotropy effects on the X/Y expected frequencies are analysed. Conclusions of this analysis, the PDF_X/Y calculation technique, and the PDF_X/Y curves obtained are useful for stereological reconstruction of anisotropically organised microcirculatory networks, with an ellipticity of their MVs being taken into consideration.     

Localization of P2X and P2Y receptors in dorsal root ganglia of the cat.

The distribution of P2X and P2Y receptor subtypes in upper lumbosacral cat dorsal root ganglia (DRG) has been investigated using immunohistochemistry. Intensity of immunoreactivity for six P2X receptors (P2X(5) receptors were immuno-negative) and the three P2Y receptors examined in cat DRG was in the order of P2Y(2) = P2Y(4)>P2X(3)>P2X(2) = P2X(7)>P2X(6)>P2X(1) = P2X(4)>P2Y(1). P2X(3), P2Y(2), and P2Y(4) receptor polyclonal antibodies stained 33.8%, 35.3%, and 47.6% of DRG neurons, respectively. Most P2Y(2), P2X(1), P2X(3), P2X(4), and P2X(6) receptor staining was detected in small- and medium-diameter neurons. However, P2Y(4), P2X(2), and P2X(7) staining was present in large- and small-diameter neurons. Double-labeling immunohistochemistry showed that 90.8%, 32.1%, and 2.4% of P2X(3) receptor-positive neurons coexpressed IB(4), CGRP, and NF200, respectively; whereas 67.4%, 41.3%, and 39.1% of P2Y(4) receptor-positive neurons coexpressed IB(4), CGRP, and NF200, respectively. A total of 18.8%, 16.6%, and 63.5% of P2Y(2) receptor-positive neurons also stained for IB(4), CGRP, and NF200, respectively. Only 30% of DRG neurons in cat were P2X(3)-immunoreactive compared with 90% in rat and in mouse. A further difference was the low expression of P2Y(1) receptors in cat DRG neurons compared with more than 80% of the neurons in rat. Many small-diameter neurons were NF200-positive in cat, again differing from rat and mouse.     

饲料营养成分对草鱼,团头鲂和青鱼鱼种阶段的饲料可消化能值的影响

为了探讨饲料可消化能值同饲料营养成分之间的关系,用Ｃｒ２Ｏ３作指示物,分别测定了鱼粉和大豆粕等饲料原料的草鱼（Ｃｔｅｎｏｐｈａｒｙｎｇｏｄｏｎ　ｉｄｅｌｌａ）团头鲂（Ｍｅｇａｌｏｂｒａｍａ ａｍｂｌｙｏｃｅｐｈａｌａ　Ｙｉｈ）青鱼（Ｍｙｌｏｐａｒｙｎｇｏｄｏｎ　ｐｉｃｅｕｓ）鱼种饲料的可消化能,用微机计算分析测试结果,发现饲料可消化能值随饲料蛋白质和／或脂肪食量增加而增加;随饲料无氮浸出物和／或纤维含量增加而降低。同时,“优选”出了有一定实用价值的估算草鱼、团头鲂和青鱼鱼种饲料可消化能值的回归方程。     

1alpha,25(OH)2D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases

Growth plate chondrocytes produce proteoglycan-rich type II collagen extracellular matrix (ECM). During cell maturation and hypertrophy, ECM is reorganized via a process regulated by 1alpha,25(OH)(2)D(3) and involving matrix metalloproteinases (MMPs), including MMP-3 and MMP-2. 1alpha,25(OH)(2)D(3) regulates MMP incorporation into matrix vesicles (MVs), where they are stored until released. Like plasma membranes (PM), MVs contain the 1alpha,25(OH)(2)D(3)-binding protein ERp60, phospholipase A(2) (PLA(2)), and caveolin-1, but appear to lack nuclear Vitamin D receptors (VDRs). Chondrocytes produce 1alpha,25(OH)(2)D(3) (10(-8)M), which binds ERp60, activating PLA(2), and resulting lysophospholipids lead to MV membrane disorganization, releasing active MMPs. MV MMP-3 activates TGF-beta1 stored in the ECM as large latent TGF-beta1 complexes, consisting of latent TGF-beta1 binding protein, latency associated peptide, and latent TGF-beta1. Others have shown that MMP-2 specifically activates TGF-beta2. TGF-beta1 regulates 1alpha,25(OH)(2)D(3)-production, providing a mechanism for local control of growth factor activation. 1alpha,25(OH)(2)D(3) activates PKCalpha in the PM via ERp60-signaling through PLA(2), lysophospholipid production, and PLCbeta. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching the MVs in PKCzeta. Direct activation of MMP-3 in MVs requires ERp60. However, when MVs are treated with 1alpha,25(OH)(2)D(3), PKCzeta activity is decreased and PKCalpha is unaffected, suggesting a more complex feedback mechanism, potentially involving MV lipid signaling.     

High lateral mobility of endogenous and transfected alkaline phosphatase: a phosphatidylinositol-anchored membrane protein

The lateral mobility of alkaline phosphatase (AP) in the plasma membrane of osteoblastic and nonosteoblastic cells was estimated by fluorescence redistribution after photobleaching in embryonic and in tumor cells, in cells that express AP naturally, and in cells transfected with an expression vector containing AP cDNA. The diffusion coefficient (D) and the mobile fraction, estimated from the percent recovery (%R), were found to be cell-type dependent ranging from (0.58 +/- 0.16) X 10(-9) cm2s-1 and 73.3 +/- 10.5 in rat osteosarcoma cells ROS 17/2.8 to (1.77 +/- 0.51) X 10(-9) cm2s-1 and 82.8 +/- 2.5 in rat osteosarcoma cells UMR106. Similar values of D greater than or equal to 10(-9) cm2s-1 with approximately 80% recovery were also found in fetal rat calvaria cells, transfected skin fibroblasts, and transfected AP-negative osteosarcoma cells ROS 25/1. These values of D are many times greater than "typical" values for membrane proteins, coming close to those of membrane lipid in fetal rat calvaria and ROS 17/2.8 cells (D = [4(-5)] X 10(-9) cm2s-1 with 75-80% recovery), estimated with the hexadecanoyl aminofluorescein probe. In all cell types, phosphatidylinositol (PI)-specific phospholipase C released 60-90% of native and transfection-expressed AP, demonstrating that, as in other tissue types, AP in these cells is anchored in the membrane via a linkage to PI. These results indicate that the transfected cells used in this study possess the machinery for AP insertion into the membrane and its binding to PI. The fast AP mobility appears to be an intrinsic property of the way the protein is anchored in the membrane, a conclusion with general implications for the understanding of the slow diffusion of other membrane proteins.     

Changes in expression of P2 receptors in rat and mouse pancreas during development and ageing

In view of the evidence for a role for extracellular ATP in both pancreatic endocrine and exocrine functions, we have investigated the expression of P2X and P2Y receptors in this tissue in neonate and aged rat and mouse. Using immunohistochemistry it was shown that P2X(1), P2X(4), P2X(7), P2Y(1) and P2Y(2) receptors were present in different regions of the rat and mouse pancreas; P2X(3) and P2X(6) receptors were not found, and P2X(5) immunolabelling was only found in some nerves. The pancreatic vasculature of both rat and mouse expressed P2X(1), P2X(2), P2Y(1) and P2Y(2) receptors in the smooth muscle. P2X(1) and P2X(4) receptors were absent in the islets of the neonate pancreas, but were progressively upregulated with age after birth. In contrast, the greatest expression of P2Y(1) in cells from the duct system was in neonate pancreas, while there was no P2Y(1) expression in aged rat pancreas. P2X(7) receptors had a consistent pattern of distribution in all of the groups examined, being located in the outer periphery of the islet. Using antibodies raised against insulin, somatostatin and glucagon, double-labelling immunofluorescence was used to identify P2X(7)-positive cells in different islet of Langerhans cell populations. Our results demonstrated a clear immunoreaction to P2X(7) receptors in islet alpha cells, while no P2X(7) was expressed in beta and delta cells. The significance of the differential expression of P2 receptors in the pancreas during development and ageing, and a possible role for the proliferation and death of the islet cell population are discussed.     

An analysis of the distribution and dose response of chromosome aberrations in human lymphocytes after in vitro exposure to137Cesium gamma radiation

The chromosome aberration yield for human lymphocytes exposed in vitro to various doses of 137Cesium has been studied. Dicentric, total acentric, and excess acentric data were seen to follow a Possion distribution. Calculated total hits demonstrated over-dispersion which could possibly be accounted for by a greater occurrence of single-hit phenomena being repaired than two-hit exchange processes. The resulting distribution generally contained an under-representation of cells with odd numbers of hits and an over-representation of zero- and even-hit classes as compared with Poisson predicted values. The relationship between dicentric yield and dose received in rads was fitted to the linear-quadratic formula Y = alpha D + beta D2 for dicentrics, yielding values of (20.1 +/- 3.8) X 10(-4) (aberrations/cell)/rad and (1.89 +/- 0.75) X 10(-6) (aberrations/cell)/rad2 for alpha and beta respectively. A plot of percent 'normal' cells versus the dose in rads resembled cell survival curves and was fitted to the relation P(D) = 100 e-Y where Y = alpha D + beta D2 with alpha = (23 +/- 11) X 10(-4) rad-1 and beta = (8.3 +/- 2.5) X 10(-6) rad-2. A possible use of scoring 'normal' cells for purposes of biological dosimetry is presented.     

GABA release by basket cells onto Purkinje cells, in rat cerebellar slices, is directly controlled by presynaptic purinergic receptors, modulating Ca2+ influx

In many brain regions, Ca(2+) influx through presynaptic P2X receptors influences GABA release from interneurones. In patch-clamp recordings of Purkinje cells (PCs) in rat cerebellar slices, broad spectrum P2 receptor antagonists, PPADS (30microM) or suramin (12microM), result in a decreased amplitude and increased failure rate of minimal evoked GABAergic synaptic currents from basket cells. The effect is mimicked by desensitizing P2X1/3-containing receptors with alpha,beta-methylene ATP. This suggests presynaptic facilitation of GABA release via P2XR-mediated Ca(2+) influx activated by endogenously released ATP. In contrast, activation of P2Y4 receptors (using UTP, 30microM, but not P2Y1 or P2Y6 receptor ligands) results in inhibition of GABA release. Immunological studies reveal the presence of most known P2Rs in >or=20% of GABAergic terminals in the cerebellum. P2X3 receptors and P2Y4 receptors occur in approximately 60% and 50% of GABAergic synaptosomes respectively and are localized presynaptically. Previous studies report that PC output is also influenced by postsynaptic purinergic receptors located on both PCs and interneurones. The high Ca(2+) permeability of the P2X receptor and the ability of ATP to influence intracellular Ca(2+) levels via P2Y receptor-mediated intracellular pathways make ATP the ideal transmitter for the multisite bidirectional modulation of the cerebellar cortical neuronal network.     

Synaptic behaviour and recombination nodules in the human XY pair

A sample of 90 XY pairs from men with normal karyotypes has been analyzed by measuring their morphological features in electron micrographs of microspread spermatocytes. The classification of human XY types (Solari, 1980) has been given stricter definitions. Stepwise splitting of the axes is seen in types 1 and 2. The development of axial branches and lenhthening of the X axis is seen in type 3. In the two subtypes a and b of type 4 the net-like filamentous array grows in length to a maximum (average=59.7 m) in subtype b. The location of the putative Y kinetochore defines a short arm that measures 22.34% of Y axis length, and the kinetochore of the X axis defines a short arm of 38.15% of the axial length. The average number of excrescences in the X axis is 19.9 and in the Y is 4.3. The frequency of a non-homologous, distal end-joining grows steadily from type 0 to type 3. The average length of the synaptonemal complex (SC) in 51 XY pairs of types 1 and 2 is 1.33 m (SD=0.65) and it corresponds to 25.54% of the Y axis length. Thus, the average SC covers the short arm of the Y and the pericentromeric region. Maximum lengths of this SC may reach up to 81.8% of the Y axis, 30 recombination nodules (RNs) were located in 26 XY pairs, and 90% of the nodules are located in the distal half of the short arm of the Y axis. Thus, RNs are restricted to a segment much shorter than the length of the average SC. A gradient of decreasing probability of recombination may reach up to the centromeric region of the Y chromosome. Some possible consequences of these facts are discussed.     

Effect of purinergic agonists and antagonists on insulin secretion from INS-1 cells (insulinoma cell line) and rat pancreatic islets

The effects of purinergic agonists on insulin release are controversial in the literature. In our studies (mainly using INS-1 cells, but also using rat pancreatic islets), ATP had a dual effect on insulin release depending on the ATP concentration: increasing insulin release (EC50 approximately/= 0.0032 microM) and inhibiting insulin release (EC50 approximately/= 0.32 microM) at both 5.6 and 8.3 mM glucose. This is compatible with the view that either two different receptors are involved, or the cells desensitize and (or) the effect of an inhibitory degradation product such as adenosine (ectonucleotidase effect) emerges. The same dual effects of ATP on insulin release were obtained using rat pancreatic islets instead of INS-1 cells. ADPbetaS, which is less degradable than ATP and rather specific for P2Y1 receptors, had a dual effect on insulin release at 8.3 mM glucose: stimulatory (EC50 approximately/= 0.02 microM) and inhibitory (EC50 approximately/= 0.32 microM). The effectiveness of this compound indicates the possible involvement of a P2Y1 receptor. 2-Methylthio-ATP exhibited an insulinotropic effect at very high concentrations (EC50 approximately/= 15 microM at 8.3 mM glucose). This indicated that distinct P2X or the P2Y1 receptor may be involved in these insulin-secreting cells. UTP increased insulin release (EC50 approximately/= 2 microM) very weakly, indicating that a P2U receptor (P2X3 or possibly a P2Y2 or P2Y4) are not likely to be involved. Suramin (50 microM) antagonized the insulinotropic effect of ATP (0.01 microM) and UTP (0.32 microM). Since suramin is not selective, the data indicated that various P2X and P2Y receptors may be involved. PPADS (100 microM), a P2X and P2Y1,4,6 receptor antagonist, was ineffective using either low or high concentrations of ATP and ADPbetaS, which combined with the suramin data hints at a P2Y receptor effect of the compounds. Adenosine inhibited insulin release in a concentration-dependent manner. DPCPX (100 microM), an adenosine (A1) receptor antagonist, inhibited the inhibitory effects of both adenosine and of high concentrations of ATP. Adenosine deaminase (1 U/mL) abolished the inhibitory effect of high ATP concentrations, indicating the involvement of the degradation product adenosine. Repetitive addition of ATP did not desensitize the stimulatory effect of ATP. U-73122 (2 microM), a PLC inhibitor, abolished the ATP effect at low concentrations. The data indicate that ATP at low concentrations is effective via P2Y receptors and the PLC-system and not via P2X receptors; it inhibits insulin release at high concentrations by being metabolized to adenosine.     

Effect of sepsis on skeletal muscle oxygen consumption and tissue oxygenation: interpreting capillary oxygen transport data using a mathematical model

Inherent in the inflammatory response to sepsis is abnormal microvascular perfusion. Maldistribution of capillary red blood cell (RBC) flow in rat skeletal muscle has been characterized by increased 1) stopped-flow capillaries, 2) capillary oxygen extraction, and 3) ratio of fast-flow to normal-flow capillaries. On the basis of experimental data for functional capillary density (FCD), RBC velocity, and hemoglobin O2 saturation during sepsis, a mathematical model was used to calculate tissue O2 consumption (Vo2), tissue Po2 (Pt) profiles, and O2 delivery by fast-flow capillaries, which could not be measured experimentally. The model describes coupled capillary and tissue O2 transport using realistic blood and tissue biophysics and three-dimensional arrays of heterogeneously spaced capillaries and was solved numerically using a previously validated scheme. While total blood flow was maintained, capillary flow distribution was varied from 60/30/10% (normal/fast/stopped) in control to 33/33/33% (normal/fast/stopped) in average sepsis (AS) and 25/25/50% (normal/fast/stopped) in extreme sepsis (ES). Simulations found approximately two- and fourfold increases in tissue Vo2 in AS and ES, respectively. Average (minimum) Pt decreased from 43 (40) mmHg in control to 34 (27) and 26 (15) mmHg in AS and ES, respectively, and clustering fast-flow capillaries (increased flow heterogeneity) reduced minimum Pt to 14.5 mmHg. Thus, although fast capillaries prevented tissue dysoxia, they did not prevent increased hypoxia as the degree of microvascular injury increased. The model predicts that decreased FCD, increased fast flow, and increased Vo2 in sepsis expose skeletal muscle to significant regions of hypoxia, which could affect local cellular and organ function.     

番茄田二代棉铃虫发生规律及药剂防治

第 2代棉铃虫Helicoverpaarmigera是危害番茄最严重的世代 ,95 %左右的卵产在番茄植株顶尖至第 4复叶层的嫩梢、嫩叶、果萼和茎基上 ,6月 2 0日至 7月 5日累计平均每株落卵 32 8粒。在番茄上第 2代棉铃虫的卵量和幼虫量与第 1代蛾量密切相关 ,卵量 (Y1)与第 1代蛾量 (x)关系式为 :Y1=-31 5 9+ 1 7783X ,n =6 ,r=0 85 6 2 ,幼虫量 (Y2 )与第 1代蛾量 (X)关系式为Y2 =31 3+ 0 1 34X ,n =6 ,r=0 86 5 2 。在第 2代棉铃虫产卵初盛期应用Bt菌剂含孢子 1 0 0亿个 mL ,2 0 0倍液喷雾 3次 ,或 1 8%阿维菌素 1 2 1 5g (a .i.) hm2 兑水喷雾 2次 ,防效显著。     

Ligand-dependent regulation of the 1,25-dihydroxyvitamin D3 receptor in rat osteosarcoma cells

The specific binding of radiolabeled 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to intact rat osteosarcoma (ROS 17/2) cells was followed for 24 h. In the presence of 0.5-1.5 nM 1,25(OH)2D3, hormone binding increased over a period of 12 h, from 1.1 X 10(4) to 1.3 X 10(5) receptors/cell. The elevated level of hormone binding persisted through 24 h provided that the initial concentration of hormone was maintained. The concentration dependence of this increase in receptor level was centered between 10 and 30 pM 1,25(OH)2D3, and the binding at 12 h exhibited the metabolite specificity expected for a 1,25(OH)2D3 receptor. The t 1/2 values for the disappearance of unoccupied and occupied receptors were roughly the same, approximately 2.7 h; therefore, the increase in hormone binding was not due to receptor stabilization. In comparison, hormone-receptor complexes appeared to dissociate with a t 1/2 of 1 h. alpha-Amanitin treatment reduced the magnitude of receptor accumulation by 50-60%, indicating that mRNA synthesis was required to achieve the maximal response. Ligand-dependent regulation of cellular receptor levels provides a mechanism for amplifying the primary hormonal signal and is predicted to influence the kinetics, magnitude, and dose dependence of cellular responses.     

Competition between oxygen and nitrate respirations in continuous culture of Pseudomonas aeruginosa performing aerobic denitrification

Continuous culture of P. aeruginosa was conducted with nitrate-containing media under the dilution rates (D) of 0.026, 0.06, and 0.13/h and the dissolved oxygen concentrations (DO) of 0-2.2 mg/L. The bacterium performed simultaneous O(2) and nitrate respiration in all of the systems studied. For each D, the (apparent) cell yield from glucose (Y(X/S)) was lower at zero DO, but did not change substantially with non-zero DO. In non-zero DO systems, Y(X/S) increased with increasing D, and when fit with a model considering cell death, gave the following parameters: maximum cell yield Y(X/S) (m) = 0.49, maintenance coefficient M(S) = 0.029 (/h), and cell decay constant k(d) = 0.014/h. The same model failed to describe the behaviors of zero-DO systems, where neither glucose nor nitrate was limiting and the limiting factor(s) remained unknown. The cell yield from accepted electron (Y(X/e)) was however relatively constant in all systems, and the energy yield per electron accepted via denitrification was estimated at approximately 69% of that via O(2) respiration. A closer examination revealed that increasing DO enhanced O(2) respiration only at extremely low DO ( <0.05 mg/L), beyond which the increasing DO only slightly increased its weak inhibition on denitrification. While O(2) was the preferred electron acceptor, the fraction of electrons accepted via denitrification increased with increasing D.     

Purinergic receptor expression in the apical plasma membrane of rat uterine epithelial cells during implantation

We examined the expression of the metabotropic P2Y(1), P2Y(2), P2Y(4), and ionotropic P2X(7) purinergic receptor subtypes in the uterine epithelium during early pregnancy in the rat. On Day 1 of pregnancy, there was no expression of P2X(7), P2Y(2), or P2Y(4) in the uterine epithelium. P2Y(1) was detected only as a diffuse label. On Day 3, P2X(7) and P2Y(2) receptor distribution was confined to the lateral plasma membranes in the epithelium. There was no expression of P2Y(4) while P2Y(1) was again detected only as a diffuse label throughout the epithelium. At the time of implantation on Day 6, a strong, continuous and area-specific P2X(7) and P2Y(2) label was noted along the entire surface of the apical epithelium suggesting a major role in calcium-modified events preceding and facilitating attachment and implantation of the blastocyst. P2Y(1) and P2Y(4) were present as a ubiquitous and nonspecific label, although the latter exhibited a minor apical deposition. These and earlier experiments with P2X subtype-specific antibodies indicate that both P2X and P2Y purinergic receptors play a role in conditioning the entire uterine epithelium for blastocyst implantation regardless of the site of attachment.     

Identification and characterization of LDL receptor gene mutations in hyperlipidemic Chinese

DNA screening for LDL receptor mutations was performed in 170 unrelated hyperlipidemic Chinese patients and two clinically diagnosed familial hypercholesterolemia patients. Two deletions (Del e3-5 and Del e6-8), eight point mutations (W-18X, D69N, R94H, E207K, C308Y, I402T, A410T, and A696G), and two polymorphisms (A370T and I602V) were identified. Of these mutations, C308Y and Del e6-8 were found in homozygosity, and D69N and C308Y were seen in unrelated patients. The effects of mutations on LDL receptor function were characterized in COS-7 cells. The LDL receptor level and activity were close to those of wild type in A696G transfected cells. A novel intermediate protein and reduction of LDL receptor activity were seen in D69N transfected cells. For R94H, E207K, C308Y, I402T, and A410T mutations, only approximately 20-64% of normal receptor activities were seen. Conversely, Del e3-5 and Del e6-8 lead to defective proteins with approximately 0-13% activity. Most of the mutant receptors were localized intracellularly, with a staining pattern resembling that of the endoplasmic reticulum and Golgi apparatus (D69N, R94H, E207K, C308Y, and I402T) or endosome/lysosome (A410T and Del e6-8). Molecular analysis of the LDL receptor gene will clearly identify the cause of the patient's hyperlipidemia and allow appropriate early treatment as well as antenatal and family studies.     

Effects of 1,25-dihydroxyvitamin D3 on cell-cycle kinetics of T 47D human breast cancer cells

The replication of several human and animal cancer cell lines is regulated in vitro and in vivo by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the hormonally active form of vitamin D3. We have examined the effects of concentrations of 1,25-(OH)2D3, which inhibit cellular replication, on the cell-cycle kinetics of a 1,25-(OH)2D3-responsive human breast cancer cell line, T 47D. After 6 or 7 days of treatment, a time period representing approximately five cell population doublings of control cultures, concentrations of 1,25-(OH)2D3 in the range 10(-9) M to 10(-6) M caused a time- and concentration-dependent decrease in cell numbers. Treatment of cells growing in charcoal-treated fetal calf serum with 10(-8) M 1,25-(OH)2D3 for 6 days reduced cell numbers to 49% +/- 9% (n = 9) of control, and this was associated with a marked increase in the proportion of cells in the G2 + M phase of the cell cycle from 9.7% +/- 0.5% (n = 11) to 19.6% +/- 2.3% (n = 9), significant by paired analysis (P less than 0.002). At higher concentrations of 1,25-(OH)2D3 (10(-7)-10(-6) M), there was a concentration-dependent decline in S phase and increases in both G0/G1 and G2 + M phase cells. Detailed analysis of the temporal changes in cell-cycle phase distribution following treatment with 2.5 X 10(-8) and 10(-7) M 1,25-(OH)2D3 showed an initial accumulation of cells in G0/G1 and depletion of S phase cells during the first 24 hr of treatment. This decline in S phase cells was not accompanied by a decline in % G2 + M indicating a transition delay in G2 or mitosis. At the lower dose these changes returned to control values at 48 hr and at later times were associated with a slight but consistent decline in G0/G1 phase and an increase in G2 + M. In contrast cells treated with 10(-7) M 1,25-(OH)2D3 had significantly elevated % G0/G1 cells at days 2 and 3, consistent with a transition delay through G1 phase. This was confirmed in stathmokinetic experiments which demonstrated an approximate sevenfold decrease in the rate of exit of cells from G0/G1 following 4 days of exposure to 10(-7) M 1,25-(OH)2D3. This accumulation of cells in G0/G1 was accompanied by a fall in % S phase cells.(ABSTRACT TRUNCATED AT 400 WORDS)