A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.

Hereditary haemochromatosis (HH), which affects some 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals of Northern European descent, results in multi-organ dysfunction caused by increased iron deposition, and is treatable if detected early. Using linkage-disequilibrium and full haplotype analysis, we have identified a 250-kilobase region more than 3 megabases telomeric of the major histocompatibility complex (MHC) that is identical-by-descent in 85% of patient chromosomes. Within this region, we have identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations. One of these is predicted to inactivate this class of proteins and was found homozygous in 83% of 178 patients. A role of this gene in haemochromatosis is supported by the frequency and nature of the major mutation and prior studies implicating MHC class I-like proteins in iron metabolism.

Spatially explicit models of divergence and genome hitchhiking.

Strong barriers to genetic exchange can exist at divergently selected loci, whereas alleles at neutral loci flow more readily between populations, thus impeding divergence and speciation in the face of gene flow. However, 'divergence hitchhiking' theory posits that divergent selection can generate large regions of differentiation around selected loci. 'Genome hitchhiking' theory suggests that selection can also cause reductions in average genome-wide rates of gene flow, resulting in widespread genomic divergence (rather than divergence only around specific selected loci). Spatial heterogeneity is ubiquitous in nature, yet previous models of genetic barriers to gene flow have explored limited combinations of spatial and selective scenarios. Using simulations of secondary contact of populations, we explore barriers to gene flow in various selective and spatial contexts in continuous, two-dimensional, spatially explicit environments. In general, the effects of hitchhiking are strongest in environments with regular spatial patterning of starkly divergent habitat types. When divergent selection is very strong, the absence of intermediate habitat types increases the effects of hitchhiking. However, when selection is moderate or weak, regular (vs. random) spatial arrangement of habitat types becomes more important than the presence of intermediate habitats per se. We also document counterintuitive processes arising from the stochastic interplay between selection, gene flow and drift. Our results indicate that generalization of results from two-deme models requires caution and increase understanding of the genomic and geographic basis of population divergence.

Space, sympatry and speciation.

Sympatric speciation remains controversial. 'Sympatry' originally meant "in the same geographical area". Recently, evolutionists have redefined 'sympatric speciation' non-spatially to require panmixia (m = 0.5) between a pair of demes before onset of reproductive isolation. Although panmixia is a suitable starting point in models of speciation, it is not a useful definition of sympatry in natural populations, because it becomes virtually impossible to find or demonstrate sympatry in nature. The newer, non-spatial definition fails to address the classical debate about whether natural selection within a geographic overlap regularly causes speciation in nature, or whether complete geographic isolation is usually required. We therefore propose a more precise spatial definition by incorporating the population genetics of dispersal (or 'cruising range'). Sympatric speciation is considerably more likely under this spatial definition than under the demic definition, because distance itself has a powerful structuring effect, even over small spatial scales comparable to dispersal. Ecological adaptation in two-dimensional space often acts as a 'magic trait' that causes pleiotropic reductions of gene flow. We provide examples from our own research.

A cell cycle analysis of growth-related genes expressed during T lymphocyte maturation.

Fetal liver or bone marrow-derived T lymphocyte precursors undergo extensive, developmentally regulated proliferation in response to inductive signals from the thymic microenvironment. We have used neonatal mouse thymocytes size-separated by centrifugal elutriation to study the cell cycle stage-specific expression of several genes associated with cell proliferation. These include genes involved in the biosynthesis of deoxyribonucleotide precursors, such as dihydrofolate reductase (DHFR), thymidylate synthase (TS), and the M1 and M2 subunits of ribonucleotide reductase, as well as c-myc, a cellular oncogene of unknown function. Using nuclear run-on assays, we observed that the transcription rates for these genes, with the exception of TS, are essentially invariant not only throughout the cell cycle in proliferating cells, but also in noncycling (G0) cells. The TS gene showed a transient increase in transcription rate in cells which bordered between a proliferating and nonproliferating status. Studies of an elutriated T cell line, S49.1, yielded similar results, indicating that the process of immortalization has not affected the transcriptional regulation of these genes. Analysis of steady-state mRNA levels using an RNase protection assay demonstrated that the levels of DHFR and TS mRNA accumulate as thymocytes progress through the cell cycle. In contrast, only the M2 subunit of ribonucleotide reductase showed cyclic regulation. Finally, in contrast to cultured cell models, we observed an abrupt fivefold increase in the steady-state level of c-myc mRNA in the transition from G1 to S-phase. We conclude from these studies that the transcriptional regulation of specific genes necessary for cellular proliferation is a minor component of the developmental modulation of the thymocyte cell cycle.

Vascular permeability factor, an endothelial cell mitogen related to PDGF.

Vascular permeability factor (VPF) is a 40-kilodalton disulfide-linked dimeric glycoprotein that is active in increasing blood vessel permeability, endothelial cell growth, and angiogenesis. These properties suggest that the expression of VPF by tumor cells could contribute to the increased neovascularization and vessel permeability that are associated with tumor vasculature. The cDNA sequence of VPF from human U937 cells was shown to code for a 189-amino acid polypeptide that is similar in structure to the B chain of platelet-derived growth factor (PDGF-B) and other PDGF-B-related proteins. The overall identity with PDGF-B is 18%. However, all eight of the cysteines in PDGF-B were found to be conserved in human VPF, an indication that the folding of the two proteins is probably similar. Clusters of basic amino acids in the COOH-terminal halves of human VPF and PDGF-B are also prevalent. Thus, VPF appears to be related to the PDGF/v-sis family of proteins.

Reset Osmostat: Facts and Controversies.

Reset osmostat (RO) consists of a change in the normal plasma osmolality threshold (reduction or increase), which consequently induces chronic dysnatremia (hyponatremia or hypernatremia). Although the early papers on RO state that hyponatremic patients with this condition are usually not symptomatic, the current view is that most patients with hyponatremia are symptomatic and should be treated. RO has been associated with a myriad of clinical conditions and has clear diagnostic criteria which are crucial to arrive at the correct diagnosis and treatment.

Asymmetric localization of a mammalian numb homolog during mouse cortical neurogenesis.

During Drosophila neurogenesis, differential segregation of Numb is necessary for daughter cells of asymmetric divisions to adopt distinct fates, at least partly by biasing the Notch-mediated cell-cell interaction. We have isolated a highly conserved mammalian homolog of Drosophila numb, m-numb. During mouse cortical neurogenesis, m-Numb is asymmetrically localized to the apical membrane of dividing ventricular neural progenitors. Depending upon the orientation of the cleavage plane, m-Numb may be distributed into one or both of the daughter cells. When expressed in Drosophila embryos, m-Numb is localized asymmetrically in dividing neural precursors and rescues the numb mutant phenotype. Furthermore, m-Numb can physically interact with mouse Notch1. We propose that some shared molecular mechanisms, both cell-intrinsic and cell-extrinsic, generate asymmetric cell divisions during neurogenesis of vertebrates and invertebrates.

Radiation and divergence in the Rhagoletis pomonella species complex: inferences from DNA sequence data.

Here, we investigate the evolutionary history and pattern of genetic divergence in the Rhagoletis pomonella (Diptera: Tephritidae) sibling species complex, a model for sympatric speciation via host plant shifting, using 11 anonymous nuclear genes and mtDNA. We report that DNA sequence results largely coincide with those of previous allozyme studies. Rhagoletis cornivora was basal in the complex, distinguished by fixed substitutions at all loci. Gene trees did not provide reciprocally monophyletic relationships among US populations of R. pomonella, R. mendax, R. zephyria and the undescribed flowering dogwood fly. However, private alleles were found for these taxa for certain loci. We discuss the implications of the results with respect to identifiable genetic signposts (stages) of speciation, the mosaic nature of genomic differentiation distinguishing formative species and a concept of speciation mode plurality involving a biogeographic contribution to sympatric speciation in the R. pomonella complex.

Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.

Vascular permeability factor (VPF) is an Mr 40-kD protein that has been purified from the conditioned medium of guinea pig line 10 tumor cells grown in vitro, and increases fluid permeability from blood vessels when injected intradermally. Addition of VPF to cultures of vascular endothelial cells in vitro unexpectedly stimulated cellular proliferation. VPF promoted the growth of new blood vessels when administered into healing rabbit bone grafts or rat corneas. The identity of the growth factor activity with VPF was established in four ways: (a) the molecular weight of the activity in preparative SDS-PAGE was the same as VPF (Mr approximately 40 kD); (b) multiple isoforms (pI greater than or equal to 8) for both VPF and the growth-promoting activity were observed; (c) a single, unique NH2-terminal amino acid sequence was obtained; (d) both growth factor and permeability-enhancing activities were immunoadsorbed using antipeptide IgG that recognized the amino terminus of VPF. Furthermore, 125I-VPF was shown to bind specifically and with high affinity to endothelial cells in vitro and could be chemically cross-linked to a high-molecular weight cell surface receptor, thus demonstrating a mechanism whereby VPF can interact directly with endothelial cells. Unlike other endothelial cell growth factors, VPF did not stimulate [3H]thymidine incorporation or promote growth of other cell types including mouse 3T3 fibroblasts or bovine smooth muscle cells. VPF, therefore, appears to be unique in its ability to specifically promote increased vascular permeability, endothelial cell growth, and angio-genesis.

A rat gene with sequence homology to the Drosophila gene hairy is rapidly induced by growth factors known to influence neuronal differentiation.

Several genes encoding transcription factors with a helix-loop-helix (HLH) motif are involved in the early process of neural development in Drosophila spp. We report the isolation from the rat a homolog of one of these genes, called hairy. The rat-hairy-like (RHL) gene is expressed early during embryogenesis. In contrast to the restricted expression of hairy mRNA in Drosophila spp., however, the mRNA encoded by RHL is detectable in all tissues examined. Stimulation of PC12 pheochromocytoma cells by nerve growth factor, basis fibroblast growth factor, or epidermal growth factor or of Rat-1 fibroblasts by epidermal growth factor causes a rapid and transient induction of the RHL gene. Thus, RHL acts as an immediate-early gene that can potentially transduce growth factor signals during the development of the mammalian embryo.

Transient inhibition of DNA synthesis results in increased dihydrofolate reductase synthesis and subsequent increased DNA content per cell.

We examined the role that blockage of cells in the cell cycle may play in the stimulation of gene amplification and enhancement of drug resistance. We found that several different inhibitors of DNA synthesis, which were each able to block cells at the G1-S-phase boundary, induced an enhanced cycloheximide-sensitive synthesis of an early S-phase cell cycle-regulated enzyme, dihydrofolate reductase, and of other proteins as well. This response was specific, in that blockage at the G2 phase did not result in overproduction of the enzyme. When the cells were released from drug inhibition, DNA synthesis resumed, resulting in a cycloheximide-sensitive elevation in DNA content per cell. We speculate that the excess DNA synthesis (which could contribute to events detectable later as gene amplification) is a consequence of the accumulation of S-phase-specific proteins in the affected cells, which may then secondarily influence the pattern of DNA replication.

A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines.

We have previously reported that rodent tumor cell lines secrete a potent vascular permeability factor with a molecular weight of 34,000-42,000 (Senger et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science (Wash. DC), 219: 983-985, 1983). This tumor-secreted vascular permeability factor (VPF) causes a rapid and completely reversible increase in microvascular permeability in the species (guinea pig or rat) from which the tumors were derived without causing mast cell degranulation or endothelial cell damage or exciting an inflammatory cell infiltrate. This VPF may be responsible, at least in part, for the increased permeability which is commonly displayed by solid and ascites tumor vessels. We have now examined 7 human tumor cell lines and have determined that 5 of them also secrete this same VPF. Antibody raised to guinea pig line 10 VPF neutralized more than 90% of the vascular permeability-increasing activity secreted by these 5 human tumor lines. Furthermore, VPFs from both guinea pig and human tumor sources bound to and were eluted similarly from immobilized heparin and comigrated identically on sodium dodecyl sulfate-polyacrylamide gels. Finally, 2 tumorigenic (in nude mice) human cell lines were found to secrete at least 14-fold more VPF than their directly matched, nontumorigenic counterparts, suggesting that elevated expression of this permeability factor may correlate with neoplastic transformation. These data suggest that a broad spectrum of tumor cells from several species, including humans, secretes a highly conserved molecule that enhances local vascular permeability and that this function may be important for tumor growth.

Purification and NH2-terminal amino acid sequence of guinea pig tumor-secreted vascular permeability factor.

Rodent and human tumor cell lines secrete a potent vascular permeability factor (VPF) which causes a rapid and substantial increase in microvascular permeability to plasma proteins without causing mast cell degranulation, or endothelial cell damage or without exciting an inflammatory cell infiltrate [D. R. Senger, S. J. Galli, A. M. Dvorak, C. A. Perruzzi, V. S. Harvey, and H. F. Dvorak. Science (Wash. DC), 219: 983-985, 1983; D. R. Senger, C. A. Perruzzi, J. Feder, and H.F. Dvorak. Cancer Res., 46: 5629-5632, 1986]. VPF now has been purified to homogeneity from guinea pig tumor cell culture medium; it is a Mr 34,000-43,000 protein, and a NH2-terminal amino acid sequence has been derived. A synthetic peptide corresponding to amino acid residues 1-24 of the native protein was used to raise rabbit antibodies which bind all of the vessel permeability-increasing activity secreted by guinea pig tumor cells and which stain purified VPF on immunoblots. These findings establish that this NH2-terminal amino acid sequence was derived from the permeability factor. Homology searches found no identity or close similarity between VPF NH2-terminal sequence and database sequences, indicating that VPF is distinct from other proteins for which sequence data are available. In particular, no sequence similarity was found between tumor-secreted VPF and other mediators of increased vessel permeability including plasma and glandular kallikreins.

Effects of propranolol and timolol on left ventricular volumes during exercise in patients with coronary artery disease.

The hemodynamic effects of beta-receptor blocking agents on the ejection fraction of patients with coronary artery disease during exercise have been studied previously using radionuclide techniques. Left ventricular volume measurements and the peak systolic pressure/end-systolic volume (PSP/ESV) index have been shown to be variables of left ventricular function that are less influenced by preload and afterload than is ejection fraction. Left ventricular volumes and PSP/ESV were therefore measured in 18 patients with proven coronary artery disease in the control state and after 2 weeks of daily maintenance therapy with either 240 mg propranolol or 60 mg timolol. Values at rest and during symptom-limited upright exercise were compared using the first pass technique and a multicrystal scintillation camera. Left ventricular volumes were measured by the area-length method. Because there was no difference between the propranolol and timolol groups, the results for both groups were combined. The ejection fraction at rest after beta-receptor blocker treatment was not significantly different from pretreatment measurements because of an increase in both end-diastolic and end-systolic volumes (p less than 0.01). However, the value for peak systolic pressure/end-systolic volume (PSP/ESV) index at rest was lower after treatment. The exercise ejection fraction was greater after treatment (p less than 0.01), owing to an increase in end-diastolic volume and unchanged end-systolic volume. In addition, there was a significant improvement in the directional change in the PSP/ESV ratio between rest and exercise from pretreatment to treatment (-1.1 +/- 2.5 to +0.2 +/- 1.2, p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Thermal properties of human IgG.

Dynamic light scattering experiments have been performed to study the aggregation kinetics of human immunoglobulin G (IgG). Aggregation and irreversible cluster growth results when IgG solutions (2-15 mg/ml) are heated above 50 degrees C. The measured scattering intensity I and effective hydrodynamic radius (R) can be described consistently by a Smoluchowski aggregation process. The number of clusters ni(t) containing i monomers at time t are computed. The radius of an i cluster is assumed to be Ri = R0 i beta, where beta is the cluster exponent. This kinetic process results in the following characteristic power law behavior: (R)/R0 = (1 + gamma R (T, C, c)t) alpha R and (I)/I0 = (1 + gamma 1 (T, C, c)t) alpha I. Here R0 = 5.51 nm, is the monomer hydrodynamic radius, and I0 the scattered intensity from the monomer solution at temperature T and concn C. A fraction, c approximately 0.48 of the IgG monomers are heat stable up to 63 degrees C and do not participate in the aggregation process. The power-law behavior of mean value of R/R0 and mean value of I/I0 indicates scaling, and indeed a very satisfactory data collapse results from our data. The best non-linear fit of the power-law forms gives alpha R = 0.48 +/- 0.05, alpha I = 1.00 +/- 0.01 and beta = 0.39 +/- 0.04. We also find that the heat aggregation of IgG is an activated process. Fits of the experimental data Gibbs free energy for the activated complex delta G* = 13.8 +/- 0.1 kcal/mole at 56 degrees C. The temp dependence of the growth rates exhibits an Arrhenius behavior with an enthalpy of activation delta H* = 120 +/- 5 kcal/mole.

Interactions of the ectodomain of HFE with the transferrin receptor are critical for iron homeostasis in cells.

Expression of wild type HFE reduces the ferritin levels of cells in culture. In this report we demonstrate that the predominant hereditary hemochromatosis mutation, C282Y(2) HFE, does not reduce ferritin expression. However, the second mutation, H63D HFE, reduces ferritin expression to a level indistinguishable from cells expressing wild type HFE. Further, two HFE cytoplasmic domain mutations engineered to disrupt potential signal transduction, S335M and Y342C, were functionally indistinguishable from wild type HFE in this assay, as was soluble HFE. These results implicate a role for the interaction of HFE with the transferrin receptor in lowering cellular ferritin levels.

Hybrid zone dynamics and species replacement between Orconectes crayfishes in a northern Wisconsin lake.

Hybrid zones that result in the genetic assimilation (replacement) of one species by another are underrepresented in the animal literature, most likely due to their transient nature. One such zone involves the rusty crayfish, Orconectes rusticus, and its congener O. propinquus. Orconectes rusticus was recently introduced into northern Wisconsin and Michigan lakes and streams, where it is hybridizing with and displacing resident O. propinquus. Here we report on a study investigating the dynamics of a hybrid zone between the two crayfish in Trout Lake, Wisconsin, where both the time (circa 1979) and location of the initial introduction are known. Our prediction was that hybridization should hasten the demise of O. propinquus because we expected that male O. rusticus (which are larger than congeners) would outcompete male O. propinquus for mates of both species. If hybrid progeny are unfit, then the result would be decreased reproductive output of O. propinquus females. However, we found a pattern of cytonuclear disequilibrium between allozymes and mtDNA suggesting that a majority (94.5%) of F1 hybrids resulted from matings between O. rusticus females and O. propinquus males. Also contrary to expectations, fecundity (O. rusticus and O. propinquus) and early hybrid survivorship did not differ significantly from nonhybrids. Moreover, adults of mixed ancestry were superior to both O. rusticus and O. propinquus in competition for a limiting food resource. Using a single-locus model, we estimated that hybridization increases the advance of O. rusticus genes in Trout Lake between 4.8% and 36.3% above that due to the previously documented ecological interactions. Consequently, whereas hybridization may be hastening the elimination of genetically pure O. propinquus, introgression is nevertheless slowing the loss of O. propinquus nuclear genes. Although our results suggest that O. rusticus and O. propinquus may not be true species under the biological concept, their ecological differences are of great conservation importance.

The hereditary hemochromatosis gene (HFE): a MHC class I-like gene that functions in the regulation of iron homeostasis.

The iron overload disorder, hereditary hemochromatosis, is one of the most common genetic diseases of individuals of Northern European descent. The disorder is characterized by the progressive accumulation of dietary iron in the major organs of the body, which if not diagnosed, leads to numerous medical maladies and eventually death. The locus for this disorder was mapped by genetic linkage to the short arm of chromosome over twenty years ago, but it was not until 1996 that the gene for this disorder was cloned by an identity-by-descent positional cloning approach. The gene, called HFE, encodes a major histocompatibility complex (MHC) class I-like protein that is mutated in approx 85% of all individuals known to have hereditary hemochromatosis (HH). Since the cloning of the HFE gene, considerable work has been carried out which has furthered our understanding of the genetics of this prevalent disorder. In addition, with the identification of the transferrin receptor as a protein capable of interacting with HFE we are now beginning to understand how a protein with the structural characteristics of an MHC class I molecule can influence cellular iron homeostasis.