Linear-scaling time-dependent density-functional theory in the linear response formalism.

We present an implementation of time-dependent density-functional theory (TDDFT) in the linear response formalism enabling the calculation of low energy optical absorption spectra for large molecules and nanostructures. The method avoids any explicit reference to canonical representations of either occupied or virtual Kohn-Sham states and thus achieves linear-scaling computational effort with system size. In contrast to conventional localised orbital formulations, where a single set of localised functions is used to span the occupied and unoccupied state manifold, we make use of two sets of in situ optimised localised orbitals, one for the occupied and one for the unoccupied space. This double representation approach avoids known problems of spanning the space of unoccupied Kohn-Sham states with a minimal set of localised orbitals optimised for the occupied space, while the in situ optimisation procedure allows for efficient calculations with a minimal number of functions. The method is applied to a number of medium sized organic molecules and a good agreement with traditional TDDFT methods is observed. Furthermore, linear scaling of computational cost with system size is demonstrated on (10,0) carbon nanotubes of different lengths.

Adenovirus-mediated retinoblastoma gene therapy suppresses spontaneous pituitary melanotroph tumors in Rb+/- mice.

The retinoblastoma gene (RB) is the prototypic tumor suppressor. Studies to date have demonstrated cancer suppression with tumor cells reconstituted with RB ex vivo and implanted into immunodeficient mice, as well as with germline transmission of a human RB transgene into tumor-prone Rb +/- mice. To mimic the therapy of cancer more closely, spontaneous pituitary melanotroph tumors arising in immunocompetent Rb +/- mice were treated with a recombinant adenovirus carrying RB cDNA. Intratumoral RB gene transfer decreased tumor cell proliferation, reestablished innervation by growth-regulatory dopaminergic neurons, inhibited the growth of tumors, and prolonged the life spans of treated animals.

Aberrant subcellular localization of BRCA1 in breast cancer.

The BRCA1 gene product was identified as a 220-kilodalton nuclear phosphoprotein in normal cells, including breast ductal epithelial cells, and in 18 of 20 tumor cell lines derived from tissues other than breast and ovary. In 16 of 17 breast and ovarian cancer lines and 17 of 17 samples of cells obtained from malignant effusions, however, BRCA1 localized mainly in cytoplasm. Absence of BRCA1 or aberrant subcellular location was also observed to a variable extent in histological sections of many breast cancer biopsies. These findings suggest that BRCA1 abnormalities may be involved in the pathogenesis of many breast cancers, sporadic as well as familial.

Knowledge and attitudes of subfertile couples towards disposition of supernumerary cryopreserved embryos: an Indian perspective.

In many cases, supernumerary embryos are cryopreserved for future use following assisted reproductive technology (ART) treatment. Once a couple has completed their family following treatment, the fate of these excess cryopreserved embryos becomes uncertain. The options available for the disposition of cryopreserved embryos are donation to other infertile couples, donation to research and discontinuation of cryostorage. In order to evaluate the knowledge and attitudes of subfertile couples from the Indian subcontinent regarding the fate of their excess cryopreserved embryos, a cross-sectional study was planned at a university-level infertility unit. A two-stage structured interview was conducted with the couples. Some questions in the interview were hypothetical in nature. In total, 87 couples were interviewed, of which 33 (37.9%) were unaware of the options for disposition of supernumerary embryos. Forty (46%) couples indicated a preference to donate their embryos to other subfertile couples, while 10 (11.5%) couples preferred donation to research. Twenty-four (27.6%) couples opted for donation to both other couples and research, while three (3.4%) couples indicated a preference to discontinue storage. Penalized bivariable logistic regression showed that none of the factors examined (i.e. age, education, income or presence of a living child) influenced the couple's decision regarding embryo donation. The majority of subfertile couples preferred to donate the embryos rather than discontinue storage. The donation of embryos to other subfertile couples was the most preferred option for disposition of embryos.

Prevention of collagen deposition following pulmonary oxygen toxicity in the rat by cis-4-hydroxy-L-proline.

Exposure of rats to high oxygen tensions causes increased collagen content of lungs and alveolar enlargement in 3-6 wk. We tested whether cis-hydroxyproline, a proline analogue that inhibits collagen synthesis, could prevent the collagen accumulation and alveolar enlargement. Rats were exposed to hyperoxia for 60 h and then to room air and hyperoxia for alternate 24-h periods for 11.5 d. Treated oxygen-exposed rats received 200 mg/kg cis-hydroxyproline twice daily over the 14-d exposure period. Control rats breathed room air. Examination of lungs on day 14 showed collagen content of oxygen-exposed lungs to be 48% greater than control (P < 0.05). The collagen content of the treated oxygen-exposed lungs was -12% of control (NS). Total lung volume was 16% greater than control in oxygen-exposed rats (P < 0.05) and 8% greater than control in treated oxygen-exposed rats (NS). Morphometric studies showed alveolar size was greater than control in oxygen-exposed rats (188+/-11 [SE] vs. 143+/-6 mumul [P < 0.05]). Oxygen-exposed, treated rats had a mean alveolar volume of 150+/-7 mumul. Lung pressure-volume curves were significantly shifted to the left of control in the oxygen-exposed rats, whereas the curves of the oxygen-exposed, treated group were identical to control. These data suggest that cis-hydroxyproline prevented the accumulation of collagen in the lungs in pulmonary oxygen toxicity. In addition, there was apparent protection from airspace dilatation and decreased lung elasticity, suggesting that alveolar enlargement after oxygen toxicity is linked to the deposition in lung tissue of new connective tissue fibers.

Pressure-induced connective tissue synthesis in pulmonary artery segments is dependent on intact endothelium.

Physiologic stimuli of connective tissue accumulation in pulmonary vascular remodeling are poorly defined. We postulated that increased pressure within central pulmonary arteries is a stimulus for connective tissue synthesis and the response is dependent on an intact endothelium. Mechanical tension equivalent to 50 mmHg pressure was applied for 4 h to isolated rat main pulmonary arteries (endothelium intact or removed), and incorporation of [14C]proline into collagen, [14C]valine into elastin, [3H]thymidine into DNA and pro alpha 1 (I) collagen mRNA levels were measured. In intact vessels, tension induced synthesis of collagen (3.1 +/- 0.4 vs. 2.3 +/- 0.5 [SEM] dpm X 10(2) [14C]-hydroxyproline/[mg protein.h]) (n = 10) and elastin (6.1 +/- 2.4 vs. 2.9 +/- 0.4 dpm X 10(3) [14C]valine/[mg protein.h]) (n = 5) (both P less than 0.05). Steady state mRNA levels of pro alpha 1 (I) collagen were also increased by tension (46 vs. 30 X 10(2) dpm hybridized/100 ng total RNA). However, the stimulus did not increase [3H]thymidine incorporation into DNA. In denuded vessels, tension had no effect on connective tissue synthesis or mRNA level of pro alpha 1 (I) collagen. Messenger RNA levels for v-sis were induced by tension in intact but not denuded vessels. Our findings establish that induction of vascular connective tissue synthesis by mechanical tension is dependent on an intact endothelium.

Mutations of N-terminal regions render the retinoblastoma protein insufficient for functions in development and tumor suppression.

To assess biological roles of the retinoblastoma protein (RB), four independent transgenic mouse lines expressing human RB with different deletions in the N-terminal region (RBdeltaN) were generated and compared with mice expressing identically regulated, full-length RB. Expression of both RB and RBdeltaN caused developmental growth retardation, but the wild-type protein was more potent. In contrast to wild-type RB, the RBdeltaN proteins were unable to rescue Rb-/- mice completely from embryonic lethality. Embryos survived until gestational day 18.5 but displayed defects in the terminal differentiation of erythrocytes, neurons, and skeletal muscle. In Rb+/- mice, expression of the RBdeltaN transgenes failed to prevent pituitary melanotroph tumors but delayed tumor formation or progression. These results strongly suggest that N-terminal regions are crucial for embryonic and postnatal development, tumor suppression, and the functional integrity of the entire RB protein. Furthermore, these transgenic mice provide models that may begin to explain human families with low-penetrance retinoblastoma and mutations in N-terminal regions of RB.

HEC, a novel nuclear protein rich in leucine heptad repeats specifically involved in mitosis.

The protein encoded by the human gene HEC (highly expressed in cancer) contains 642 amino acids and a long series of leucine heptad repeats at its C-terminal region. HEC protein is expressed most abundantly in the S and M phases of rapidly dividing cells but not in terminal differentiated cells. It localizes to the nuclei of interphase cells, and a portion distributes to centromeres during M phase. Inactivation of HEC by microinjection of specific monoclonal antibodies into cells during interphase severely disturbs the subsequent mitoses. Disordered sister chromatid alignment and separation, as well as the formation of nonviable cells with multiple, fragmented micronuclei, are common features observed. These results suggest that the HEC protein may play an important role in chromosome segregation during M phase.

Retinoblastoma protein enhances the fidelity of chromosome segregation mediated by hsHec1p.

Retinoblastoma protein (Rb) plays important roles in cell cycle progression and cellular differentiation. It may also participate in M phase events, although heretofore only circumstantial evidence has suggested such involvement. Here we show that Rb interacts, through an IxCxE motif and specifically during G(2)/M phase, with hsHec1p, a protein essential for proper chromosome segregation. The interaction between Rb and hsHec1p was reconstituted in a yeast strain in which human hsHEC1 rescues the null mutation of scHEC1. Expression of Rb reduced chromosome segregation errors fivefold in yeast cells sustained by a temperature-sensitive (ts) hshec1-113 allele and enhanced the ability of wild-type hsHec1p to suppress lethality caused by a ts smc1 mutation. The interaction between Hec1p and Smc1p was important for the specific DNA-binding activity of Smc1p. Expression of Rb restored part of the inactivated function of hshec1-113p and thereby increased the DNA-binding activity of Smc1p. Rb thus increased the fidelity of chromosome segregation mediated by hsHec1p in a heterologous yeast system.

A new member of the hsp90 family of molecular chaperones interacts with the retinoblastoma protein during mitosis and after heat shock.

A gene encoding a new heat shock protein that may function as a molecular chaperone for the retinoblastoma protein (Rb) was characterized. The cDNA fragment was isolated by using the yeast two-hybrid system and Rb as bait. The open reading frame of the longest cDNA codes for a protein with substantial sequence homology to members of the hsp90 family. Antibodies prepared against fusions between glutathione S-transferase and portions of this new heat shock protein specifically recognized a 75-kDa cellular protein, hereafter designated hsp75, which is expressed ubiquitously and located in the cytoplasm. A unique LxCxE motif in hsp75, but not in other hsp90 family members, appears to be important for binding to the simian virus 40 T-antigen-binding domain of hypophosphorylated Rb, since a single mutation changing the cysteine to methionine abolishes the binding. In mammalian cells, Rb formed complexes with hsp75 under two special physiological conditions: (i) during M phase, when the envelope that separates the nuclear and cytoplasmic compartments broke down, and (ii) after heat shock, when hsp75 moved from its normal cytoplasmic location into the nucleus. In vitro, hsp75 had a biochemical activity to refold denatured Rb into its native conformation. Taken together, these results suggest that Rb may be a physiological substrate for the hsp75 chaperone molecule. The discovery of a heat shock protein that chaperones Rb identifies a mechanism, in addition to phosphorylation, by which Rb is regulated in response to progression of the cell cycle and to external stimuli.

Earlier onset of melanotroph carcinogenesis in mice with inherited mutant paternal allele of the retinoblastoma gene.

The role of genomic imprinting in the development of tumors with defective retinoblastoma protein function remains debatable. Disruption of either parental allele of the murine retinoblastoma (Rb) gene is sufficient for spontaneous melanotroph carcinogenesis to occur in almost all Rb+/- mice. Nevertheless, mice with a disrupted paternal Rb allele succumb to tumors faster. In these animals, the first foci of proliferating atypical Rb-negative cells appear and progress to overtly malignant tumors earlier. In addition, more foci of early atypical proliferation are observed. In Rb+/- mice, however, parental origin influences neither Rb expression nor proliferation of melanotrophs. Accordingly, Rb-/- mice rescued by the human RB transgene transmitted either paternally or maternally have similar survival rates. Taken together, the data point to the existence of an imprinted gene in an Rb-linked locus. The function of this gene affects the onset of melanotroph carcinogenesis, likely by controlling preferential survival of the cells with secondary loss of the Rb maternal allele. Rb+/- mice may serve as useful models to identify and characterize genomic imprinting mechanisms influencing carcinogenesis associated with Rb loss of function.

Susceptibility to tumors induced in mice by ethylnitrosourea is independent of retinoblastoma gene dosage.

The retinoblastoma gene (RB) is a classical tumor suppressor. Several studies have shown that RB dosage is important in determining biological effects. To explore the effect of RB dosage on susceptibility to cancer, three groups of congenic C57BL/6 mice, each of which expresses a different amount of Rb protein from one, two, or three alleles, were treated at postnatal day 12 with a single 60-mg/kg body weight i.p. dose of the DNA-alkylating agent N-ethyl-N'-nitrosourea (ENU). Mice heterozygous for the RB gene developed characteristic pituitary tumors with nearly complete penetrance, whether or not they were treated with ENU. Tumors initiated earlier or progressed more rapidly, however, in ENU-treated mice. Furthermore, although mice treated with ENU had a higher incidence of several nonpituitary tumors compared with untreated controls, no significant differences in the incidence of these tumors were found between wild-type mice (mRB+/+), mice carrying only one normal RB allele and deficient in Rb protein expression (mRB+/-), and mice overexpressing Rb protein from two normal murine RB alleles and a human RB transgene (mRB+/+, hRB+/-). These studies underscore the tissue and mechanistic specificity of tumor predisposition caused by an inherited 50% reduction in RB dosage and indicate that most ENU-induced tumors occur independent of RB inactivation. Nonetheless, they suggest that certain point mutations induced by ENU may participate in the sequence of molecular steps involved in progression of tumor-prone, RB-deficient cells to the fully malignant state.

Chemotactic activity of collagen-like polypeptides for human peripheral blood neutrophils.

Damage to interstitial connective tissue is associated with the rapid accumulation of monocytes and neutrophils at the site of injury. To study the role of collagen fragments in neutrophil migration, we analyzed the chemotactic properties of peptide fragments of bovine collagen digested with bacterial collagenase or cyanogen bromide and small molecular weight synthetic polypeptides containing proline (Pro), hydroxyproline (Hyp), and glycine (Gly), the major amino acids that comprise collagen. Using the Boyden chamber and under agarose techniques, we found that collagen fragments were as potent in inducing chemotaxis in neutrophils as the bacterial-derived peptide f-met-leu-phe. The synthetic polytripeptides (Pro-Pro-Gly)5 and (Pro-Hyp-Gly)5 were found to be equipotent in inducing chemotaxis, producing a maximal induction of chemotaxis at 5-10 nM. This suggests that Hyp, the unique imino acid found in collagen, is not required for chemotactic activity. Increasing the length of the synthetic tripeptide from 5 to 10 subunits decreased its chemotactic activity, while the single tripeptide subunit (Pro-Hyp-Gly)1 was the least active peptide, inducing a maximal response at 100 nM. To study the structural requirements for chemotaxis, Pro-Hyp-Gly tripeptides were synthesized with modifications at the N and C terminals ends. Addition of a methyl group to the carboxyl of Gly to form an ester enhanced the chemotactic activity of the peptide by 50%, while substitutions on the amino terminus with an acetyl group decreased the chemotactic activity by 50%. Substitution on the amino terminus with a Boc group decreased the chemotactic activity by 100%. These results indicate that there are specific structural requirements for chemotaxis induced by peptides having a collagen-like sequence of amino acids.

Circulating fibroblast growth factor-like autoantibodies in two patients with multiple endocrine neoplasia type 1 and prolactinoma.

Basic fibroblast growth factor (bFGF) is a potent endothelial cell mitogen found in a variety of normal and tumor tissues. bFGF lacks a classical amino-terminal signal sequence and is not readily detectable in plasma from normal subjects. In earlier studies we showed increased bFGF-like mitogenic activity for parathyroid-derived endothelial cells and (increased) bFGF immunoreactivity (0.24-1.28 ng/mL) in plasma of subjects with multiple endocrine neoplasia type 1 (MEN-1). In the present study we examined the proliferative activity of MEN-1 and normal plasmas (applied to protein-A columns) in calf pulmonary artery endothelial cells. Protein-A-eluted activity in plasma from MEN-1 prolactinoma plasma exceeded activity from normal and MEN-1 nonprolactinoma plasma in three of eight MEN-1 subjects with untreated or recurrent prolactinoma. Protein-A-eluted active fractions from MEN-1 prolactinoma plasma had several properties of an immunoglobulin G, including affinity for antihuman immunoglobulin G (IgG) agarose, sensitivity to thiols, and (prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions) apparent mol wt corresponding to those of the heavy and light chains of IgG. The IgG fraction of MEN-1 prolactinoma plasma had far more activity in endothelial cells than did optimal concentrations of known growth factors or conditioned medium from prolactinoma cells. Endothelial cell bioactivity in protein-A-eluted fractions from MEN-1 prolactinoma plasma was neutralized 70% by rabbit antibodies to intact bFGF. These results imply novel growth stimulatory bFGF-like autoantibodies in a subset of MEN-1 patients with prolactinoma.

The retinoblastoma protein as a fundamental mediator of growth and differentiation signals.

The retinoblastoma gene (RB) is the prototype of the tumor suppressor genes, which play critical roles in the genesis of cancer in humans. Mouse models created through gene knock-out and transgenic methods were established for exploring and manipulating RB in vivo. These models and several other pieces of evidence have shown that the retinoblastoma protein (Rb) plays dual roles in gating cell cycle progression and promoting cellular differentiation. The molecular mechanisms involved in these roles are becoming more obvious in some biological systems: Rb sequesters the transcription factor of E2F to regulate entry of cell cycle but enhances the activities of another class of the transcription factors, exemplified by NF-IL6, to initiate terminal cellular differentiation. Thus, the Rb protein can serve as a mediator for extracellular signals of growth or differentiation. The fundamental question of why only limited cell types are susceptible to tumor formation when Rb expression is lost, however, remains unanswered at present.

Blunted respiratory drive in congenital myopathy.

Two patients with clinically mild congenital myopathies presented with chronic respiratory failure. Muscle weakness alone could not account for the respiratory insufficiency since static respiratory pressures were not markedly impaired, ventilation during exercise was normal, and daytime ventilation was normal if ventilatory assistance was provided at night. The ventilatory responses to inhaled carbon dioxide were very low, suggesting that impairment of the central nervous respiratory chemoreceptor contributed to hypoventilation. These patients and others described in the literature suggest that central depression of ventilation may occur more frequently than previously recognized in patients with muscular disorders. Patients with chronic respiratory failure due to central depression of respiratory drive can be effectively managed by assisted ventilation at night.

Kidney transplantation during the first trimester of pregnancy: immunosuppression with mycophenolate mofetil, tacrolimus, and prednisone.

We present a case of living, related-donor kidney transplantation during the first trimester of pregnancy. The patient received mycophenolate mofetil (MMF), tacrolimus, and prednisone throughout the entire pregnancy. This is the first reported case of use of MMF during pregnancy. The mother did well, except for mild preeclampsia and mild renal insufficiency at term. The baby girl was born prematurely at week 353/7. The only possible teratogenic effects detected included hypoplastic nails and short fifth fingers. No chromosomal abnormalities were found. The child is growing and developing normally. Although we do not recommend the use of mycophenolate mofetil during pregnancy based on this experience, it is reassuring to know that a successful outcome can be expected in mothers treated with MMF during pregnancy.

Shared cadaver donor-husband HLA class I mismatches as a risk factor for renal graft rejection in previously pregnant women.

During the last few years, we have observed four cases in which accelerated rejection of a cadaver donor kidney in a previously pregnant woman could be clearly attributed to the rapid emergence of anti-human leukocyte antigen (HLA) antibodies that had been stimulated by mismatched paternal antigens but were completely undetectable at the time of transplantation. In addition to reviewing those cases, we also reviewed data on 19 other women with a history of at least one pregnancy who underwent transplantation with a first cadaveric kidney since 1991 and were followed for at least six months. The HLA antigens of the husbands had to have been determined and all accelerated rejection or early graft losses due to confirmed or presumed immunological causes were considered. Of the 19 additional women meeting these inclusion criteria, three suffered early immunological graft loss. As in our index cases, two of these women had also received kidneys from donors who shared at least one major immunogenic mismatched antigen with the respective husband for a total of six of seven women with early immunological graft loss. Only one of the 16 women without accelerated rejection or early immunological graft loss had a donor who shared a mismatched antigen with her husband. The difference between the two groups is statistically significant (p = 0.0005). These findings, considered with individual cases reported by other groups, indicate that transplantation from a cadaver donor with immunogenic mismatched class I HLA antigen(s) shared with the husband should be avoided in women with a previous history of pregnancy even when anti-HLA antibodies are not currently detected.

Strain specific respiratory air space enlargement in aged rats.

We studied lung structure and function in Fischer-344 and Sprague Dawley rats to compare the pathophysiologic features of the aged lung in animal strains. Both strains were maintained under identical conditions of minimal exposure to injurious environmental agents. We measured the number, size, and surface area of alveoli, pressure-volume characteristics and connective tissue content of lungs at midlife (12 or 14 months of age) and old age (24 months of age). Results showed differences in the older versus younger group of the Sprague Dawley strain as indicated by enlarged air spaces [154 +/- 21 (SEM) versus 118 +/- 13 micromicroliter] (p less than 0.05), increased collagen (hydroxyproline content 4.1 +/- 0.1 versus 3.0 +/- 0.1 mg/lung) (p less than 0.05), and a leftward shifted pressure-volume curve. There was no change in surface area or alveolar number. The structural lesions are consistent with air space enlargement with fibrosis and not emphysema. In contrast, no major changes were found in the lungs with age in Fischer-344 rats. We hypothesize that in the Sprague Dawley strain the aging process impairs the ability of the lung to maintain normal structure and function. Two strains of rats which differ pathologically in old age may be useful in the study of the effects of aging on the lung.

The potential of gene therapy for treatment of kidney diseases.

As knowledge of the genes and molecular events involved in specific kidney disorders expands, so do the potential applications for treating the disorders on fundamental levels. Progress in gene therapy for genetic diseases for which molecular events are simpler or better characterized has already been substantial. In addition, exciting first steps in targeting genes specifically to the kidney have emerged, although much work in defining kidney-specific genes and gene regulatory elements still needs to be done. To provide a framework for understanding the concepts and problems involved in gene therapy, we will discuss basic aspects of gene structure and regulation, gene delivery vectors, steps necessary to achieve tissue- and cell-specific expression of delivered genes, and some present and future applications of gene therapy in kidney diseases.