Late Holocene climate and cultural changes in the southwestern United States.

Columnar stalagmites in caves of the Guadalupe Mountains during the late Holocene record a 4000-year annually resolved climate history for the southwestern United States. Annual banding, hiatuses, and high-precision uranium-series dating show a present day-like climate from 4000 to 3000 years ago, following a drier middle Holocene. A distinctly wetter and cooler period from 3000 to 800 years ago was followed by a period of present day-like conditions, with the exception of a slightly wetter interval from 440 to 290 years before the present. The stalagmite record correlates well with the archaeological record of changes in cultural activities of indigenous people. Such climate change may help to explain evidence of dwelling abandonment and population redistribution.

Melting of the Earth's lithospheric mantle inferred from protactinium-thorium-uranium isotopic data

The processes responsible for the generation of partial melt in the Earth's lithospheric mantle and the movement of this melt to the Earth's surface remain enigmatic, owing to the perceived difficulties in generating large-degree partial melts at depth and in transporting small-degree melts through a static lithosphere. Here we present a method of placing constraints on melting in the lithospheric mantle using 231Pa-235U data obtained from continental basalts in the southwestern United States and Mexico. Combined with 230Th-238U data, the 231Pa-235U data allow us to constrain the source mineralogy and thus the depth of melting of these basalts. Our analysis indicates that it is possible to transport small melt fractions--of the order of 0.1%--through the lithosphere, as might result from the coalescence of melt by compaction owing to melting-induced deformation. The large observed 231Pa excesses require that the timescale of melt generation and transport within the lithosphere is small compared to the half-life of 231Pa (approximately 32.7 kyr). The 231Pa-230Th data also constrain the thorium and uranium distribution coefficients for clinopyroxene in the source regions of these basalts to be within 2% of one another, indicating that in this setting 230Th excesses are not expected during melting at depths shallower than 85 km.

Reversal of cathepsin D routing modulation in MCF-7 breast cancer cells expressing antisense insulin-like growth factor II (IGF-II).

We previously demonstrated that expression of IGF-II modulates the routing of cathepsin D in MCF-7 cells. In our present study, we transfected antisense IGF-II into IGF-II secreting MCF-7 cells to test the hypothesis that blocking IGF-II may reduce the secretion of cathepsin D in breast cancer cells. The concentration of IGF-II in media conditioned by the antisense clone was reduced to almost undetectable levels. Likewise, Northern blotting analysis revealed that IGF-II mRNA was nearly undetectable in the antisense transfected cells. Metabolic labeling experiments performed with 10 mM mannose 6-phosphate present in the medium to block reuptake of lysosomal enzymes demonstrated that cathepsin D secretion was dramatically reduced. Similarly, a significant reduction in cathepsin D was observed when conditioned media and cell extracts were examined by Western blotting after a 48 h incubation. No changes in cathepsin D mRNA in antisense cells were detected by Northern blot analysis. We conclude that endogenous IGF-II may modulate the routing of cathepsin D by interfering with receptor trafficking in MCF-7 cells, and that this modulation is reversible. Abnormally high levels of IGF-II may alter this homeostasis, conferring on breast cancer cells an advantageous mechanism that promotes rapid growth, and may facilitate metastasis.

Quantification of insulin-like growth factor I (IGF-I) without interference by IGF binding proteins.

A chemiluminescent dot blot assay has been developed by our laboratory for rapid determinations of IGF-I in serum-free conditioned media (CM) collected from cultured cells. In contrast to IGF-I radioimmunoassays (RIAs), the IGF binding proteins (IGFBPs) did not interfere with the dot blot assay and did not require the laborious (and sometimes ineffective) removal of IGFBPs. Although all six IGFBPs were shown to bind to 125I IGF-I, none interfered with IGF-I detection on nitrocellulose dot blots. In contrast, an RIA using the same Oncogene monoclonal antibody (clone 82-9A) showed interference by IGFBP-1, IGFBP-2, and IGFBP-4. The IGF-I dot blot assay was sensitive (0.125-8.0 ng IGF-I), specific (assay crossreactivity with IGF-II is less than 1%), and reproducible (intra-assay variance < or = 6%; inter-assay variance < 12%) when chemiluminescence was quantified by phosphorimager and Molecular Analyst software (BioRad). The apparent sensitivity of the enhanced chemiluminescence (ECL) reagent to serum, precludes the use of this assay for IGF-I determination in serum or in serum-containing media.

Insulin-like growth factor II modulates the routing of cathepsin D in MCF-7 breast cancer cells.

A previous observation that insulin-like growth factor II (IGF-II) inhibits the cellular uptake of a lysosomal enzyme by inhibiting binding to the IGF-II/mannose 6-phosphate receptor led to the proposal that, in a cell producing IGF-II, the routing of lysosomal enzymes might be altered. To test this hypothesis MCF-7 breast cancer cells were transfected with pRc/CMV vector only (CMV) or vector containing IGF-II complementary DNA encoding either mature (M-II) or precursor (P-II) IGF-II, and the routing of cathepsin D, a predominant lysosomal enzyme in this cell line, was examined. The concentration of IGF-II in media conditioned by P-II clones (11.2 +/- 4.3 micrograms/ml) was much higher than in media conditioned by M-II clones (1.3 +/- 1.5 micrograms/ml). Metabolic labeling experiments were performed with 10 mM mannose 6-phosphate present in the medium to block reuptake of lysosomal enzymes. Cell extracts (C) and media (M) were immuno-precipitated with a cathepsin D antiserum, and immunoprecipitates were analyzed by SDS-PAGE. The mean of the C/M ratio of cathepsin D for the seven P-II clones (1.60 +/- 0.13) was significantly lower than for the six CMV clones (3.47 +/- 0.48). Similar results were obtained when conditioned M and C were examined by immunoblotting after a 48-h incubation. The mean of the C/M ratio for the seven P-II clones (11.4 +/- 1.6) was significantly lower than for the six CMV clones (24.9 +/- 5.2). There was also a strong negative correlation between the ratio of intracellular cathepsin D to extracellular cathepsin D and relative cathepsin D synthesis (r = 0.843), consistent with increased cathepsin D production in cells overexpressing IGF-II. It is concluded that endogenous IGF-II modulates the routing of cathepsin D in MCF-7 cells.

Strontium isotopic variations of Neoproterozoic seawater: implications for crustal evolution.

We report high precision Sr isotopic data on carbonates from the Neoproterozoic Shaler Group, Victoria Island, Northwest Territories, Canada. Lithostratigraphic correlations with the relatively well-dated Mackenzie Mountains Supergroup constrain Shaler deposition to approximately 770-880 Ma, a range corroborated by 723 +/- 3 Ma lavas that disconformably overlie Shaler carbonates and by Late Riphean microfossils within the section. Samples with low 87Rb/86Sr ratios (<0.01) were selected for Sr isotopic analysis. Delta 18O, Mn, Ca, Mg, and Sr data were used to recognize altered samples. The altered samples are characterized by high Mn/Sr (> or = 2) and variable delta 18O; most are dolomites. The data indicate that between ca. 790-850 Ma the 87Sr/86Sr ratio of seawater varied between 0.70676 and 0.70561. The samples show smooth and systematic variation, with the lowest 87Sr/86Sr value of 0.70561 at ca. 830 Ma. The low 87Sr/86Sr ratio of carbonates from the lower parts of our section is similar to a value reported for one sample from the Adrar of Mauritania (approximately 900 Ma), West African Craton. Isotopic ratios from the upper part of the Shaler section are identical to values from the lower part of the Neoproterozoic Akademikerbreen Group, Spitsbergen. Although a paucity of absolute age determinations hinders attempts at the precise correlation of Neoproterozoic successions, it is possible to draw a broad outline of the Sr isotopic composition of seawater for this period. Indeed, the Sr isotope data themselves provide a stratigraphic tool of considerable potential. Data from this study and the literature are used to construct a curve of the 87Sr/86Sr ratio of Neoproterozoic seawater. The new data reported in this study substantially improve the isotopic record of Sr in seawater for the period 790-850 Ma. The Sr isotope composition of seawater reflects primarily the balance between continental Sr input through river input and mantle input via hydrothermal circulation of seawater through mid-ocean ridges. Coupling of Nd and Sr isotopic systems allows us to model changes in seafloor spreading rates (or hydrothermal flux) and continental erosion. The Sr hydrothermal flux and the erosion rate (relative to present-day value) are modeled for the period 500-900 Ma. The results indicate that the hydrothermal flux reached a maximum value at ca. 830 Ma. In contrast, a large peak in erosion rate is indicated at ca. 570 Ma. The peaks in hydrothermal flux and erosion rate are most likely related to developments in the Pan-African and related orogenic events, whose initial development is characterized by production of juvenile crust during supercontinental break up and rifting. The time ca. 570 Ma is characterized by continent-continent collision and production of recycled crust. Sr isotope data from Proterozoic carbonates offer a valuable resource for understanding large-scale crust dynamics.

Rapid HPLC techniques for globin chain synthesis studies.

Globin chain synthesis and alpha/beta ratios were determined in a number of normal subjects, alpha-thalassemia-2 homozygotes, and beta-thalassemia trait using three different techniques. Cation exchange high performance liquid chromatography on a Pharmacia Mono-S, HR 5/5 and reversed phase high performance liquid chromatography on a semi-preparative Vydac C4 column were compared with the traditional carboxymethylcellulose chromatography. Both high performance liquid chromatography columns give excellent results when 2 mg of hemoglobin was chromatographed in each analysis. By modifying the protocols for column equilibration and gradient shape for preparative Vydac C4 columns, conditions were found yielding excellent resolutions of the labeled globin chains in less than an hour without the need for substantial increase of the flowrate. This method was found to be superior to other methods and may be a suitable alternative for the classical carboxymethylcellulose chromatography. Up to five specimens could easily be analyzed in a single day with this system.

Hemoglobin Pasadena: identification of the gene mutant by DNA analysis using synthetic DNA probes.

Hemoglobin Pasadena [beta 75(E19)Leu----Arg] was found in a boy who had an acute episode of anemia and rapid splenic enlargement. His father was the only other member of a large family with this hemoglobinopathy. We have used gene mapping techniques for direct identification of the beta-globin gene mutation. To correlate the DNA findings with the structural identification of this variant, we have also performed globin chain separation and analysis of the tryptic peptides using high performance liquid chromatography and secondary ion mass spectral analysis.

Reverse phase high-performance liquid chromatography and secondary ion mass spectrometry. A strategy for identification of ten human hemoglobin variants.

Ten abnormal hemoglobins were detected and characterized in individual cases referred to our laboratory for evaluation of hematological problems. Six of these variants were electrophoretically silent and could be detected by reverse phase high-performance liquid chromatography (HPLC) analysis. HPLC was also used to analyze the tryptic peptides of each individual variant. In most of these variants, secondary ion mass spectra of the mixture of the tryptic peptides could reveal the aberrant peptide and predict possible substitution through the mass difference between the normal and abnormal peptide. The mass spectra of the isolated abnormal peptide generally contained sufficient fragment ions to define the position of the amino acid substitution, obviating the need for lengthy sequencing procedures. Combination of the two techniques.

Hemoglobin North Chicago (beta 36 [C2] proline----serine): a new high affinity hemoglobin.

Hemoglobin North Chicago, beta 36 [C2] Pro----Ser is a new high oxygen affinity hemoglobin variant. It was discovered in a 52-year-old male with erythrocytosis since age 20 who had been treated with different regimens for polycythemia vera including several courses of 32P. The variant is electrophoretically silent with normal stability and increased oxygen affinity (P50 16.6 mm Hg at 37 degrees C, pH 7.4). Characterization of the structure of hemoglobin North Chicago involved the use of HPLC, secondary ion mass spectral analysis of the tryptic peptides and conventional fingerprinting. Hemoglobin North Chicago manifested bizarre hydrophobicity of its beta-chains, as demonstrated by reverse phase HPLC and Triton X-100 electrophoresis. This behavior is not expected from the substitution of proline to serine. Proline residue beta 36 [C2] is one of the invariant residues of the beta-chains of all known mammals and most vertebrates. This residue is involved in the alpha 1 beta 2 contacts of hemoglobin molecule and its substitution to serine is possibly associated with conformational changes and alteration of hemoglobin function.

A silent hemoglobin variant detected by HPLC: hemoglobin City of Hope beta 69 (E13) Gly----Ser.

A silent hemoglobin variant with substitution of serine for glycine at position 69 of the beta-chain was discovered in a healthy individual. Reverse-phase HPLC was used for globin chain separation and to separate the tryptic peptides of the variant. This variant was undetectable by conventional methods of protein separation such as electrophoresis, isoelectric focusing, and ion-exchange chromatography. This observation demonstrates the potential of reverse-phase HPLC as a tool for the search and detection of neutral substitutions in variants of hemoglobin and other proteins, and its usefulness for screening genetic variations in human populations.