Hormone measures in finger-prick blood spot samples: New field methods for reproductive endocrinology

Comparative endocrine studies have notably advanced understanding of ecological factors that contribute to variation in human reproductive function. Such research has relied on methodological advances that permit hormone determinations in samples that are easily and safely collected, stored, and transported, most recently on measurement of steroids in saliva. This report seeks to further expand the scope of endocrine research by demonstrating the value of blood spot samples collected by finger prick. As a sampling strategy, finger-prick blood spot collection offers the advantages of short collection time, low invasiveness, repeatability, absence of postcollection processing, low biohazard risk, and ease of sample storage and transport. We document good sample stability and present sensitive assay methods for a range of steroids and proteins (FSH, LH, PRL, T, E2, DHEAS, androstenedione, cortisol, SHGB) in blood spots that require sample volumes of 3–12 μl and display good reliability, specificity, precision, accuracy, and convertibility of results to plasma/serum equivalent concentrations. Laboratory evaluation was augmented by a feasibility study at a remote site in Papua New Guinea that confirmed validity and stability of blood spot collections under field conditions. Research applications of blood spot sampling are illustrated with a series of studies, including cross-sectional surveys for developmental and life span endocrinology, a longitudinal, population-based developmental epidemiologic study of puberty, and serial sampling in a dynamic study of neuroendocrine response to suckling. We conclude that the sampling features and wide range of measurable biomolecules of blood spots do constitute a methodological advance for endocrine research. Am J Phys Anthropol 104:1–21, 1997. © 1997 Wiley-Liss, Inc.     

Neurotrophin-3- and norepinephrine-mediated adrenergic differentiation and the inhibitory action of desipramine and cocaine

In the presence of neurotrophin-3 (NT-3), high-affinity norepinephrine (NE) uptake by quail neural crest cells was significantly increased as judged by in vitro colony assay of adrenergic differentiation. In the presence of the related neurotrophins nerve growth factor (NGF) or brain-derived neurotrophic (BDNF) factor, or of basic fibroblast growth factor (bFGF), there were no significant changes. When NE was added to the culture medium in addition to NT-3, more colonies contained dopamine-β-hydroxylase (DBH)-immunoreactive cells, an enzyme that is characteristic for adrenergic cells. The NE-mediated increase in the portion of colonies that contained DBH-immunoreactive cells was prevented by the tricyclic antidepressant desipramine (DMI) and by cocaine, two types of drug that block cellular transport of NE. To further examine whether NE acts via uptake, colony assays were performed in the presence and absence of adrenergic antagonists and agonists. These would be expected to mimic the DMI and NE effects, respectively, if the mechanism of action involved activation of adrenergic autoreceptors. Neither class of drug showed a detectable effect within a wide range of concentrations. Immunocytochemistry using antibodies against β1 and β2 adrenergic receptors further supported the notion that DMI action and β-receptor expression are not causally related. Ratio imaging was subsequently used in an attempt to elucidate the mechanism of NE action. Within a few minutes of addition of NE to the culture medium, there was an increase in intracellular free calcium in a subset of neural crest cells. Taken together, our data indicate that NT-3 is involved in the appearance of the NE transporter (NET) during embryonic development; internalized NE directly or indirectly increases adrenergic differentiation as measured by immunoreactivity of the adrenergic biosynthetic enzyme DBH; and norepinephrine uptake inhibitors have teratogenic potential. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 262–280, 1997.     

Crystal structure of a recombinant form of the maltodextrin-binding protein carrying an inserted sequence of a B-cell epitope from the preS2 region of hepatitis B virus

We report the crystal structure of MalE-B133, a recombinant form of the maltodextrin-binding protein (MBP) of Escherichia coli carrying an inserted amino-acid sequence of a B-cell epitope from the preS2 region of the hepatitis B virus (HBV). The structure was determined by molecular replacement methods and refined to 2.7 Å resolution. MalE-B133 is an insertion/deletion mutant of MBP in which residues from positions 134 to 142, an external α helix in the wild-type structure, are replaced by a foreign peptide segment of 19 amino acids. The inserted residues correspond to the preS2 sequence from positions 132 to 145 and five flanking residues that arise from the creation of restriction sites. The conformation of the recombinant protein, excluding the inserted segment, closely resembles that of wild-type MBP in the closed maltose-bound form. MalE-B133 was shown by previous studies to display certain immunogenic and antigenic properties of the hepatitis B surface antigen (HBsAg), which contains the preS2 region. The crystal structure reveals the conformation of the first nine epitope residues (preS2 positions 132 to 140) exposed on the surface of the molecule. The remaining five epitope residues (preS2 positions 141 to 145) are not visible in electron density maps. The path of the polypeptide chain in the visible portion of the insert differs from that of the deleted segment in the structure of wild-type MBP, displaying a helical conformation at positions 134 to 140 (preS2 sequence numbering). A tripeptide (Asp-Pro-Arg) at the N terminus of the helix forms a stable structural motif that may be implicated in the cross-reactivity of anti-HBsAg antibodies with the hybrid protein. Proteins 27:1–8 © 1997 Wiley-Liss, Inc.     

Parathyroid hormone uses both adenylate cyclase and protein kinase C to regulate acid production in osteoclasts

Osteoclasts, isolated from the endosteum of 2.5- to 3-week-old chickens, were treated with acridine orange, a hydrogen ion concentration-sensitive fluorescent dye, in order to monitor changes in acid production. The adenylate cyclase inhibitor, alloxan, blocked parathyroid hormone (PTH)-stimulated acid production. Dibutyryl cyclic adenosine monophosphate, a membrane-permeant form of cyclic adenosine monophosphate, mimicked the PTH effect. Bisindolylmaleimide, a specific inhibitor of protein kinase C (PKC), blocked the initial stimulation (15, 30, and 60 min) of acid production by PTH but had no effect on long-term stimulation (120 min). Confocal microscopy of osteoclasts stained with fluorescein-conjugated bisindolylmaleimide revealed a shift in location of PKC from the cytoplasm to the plasma membrane region after treatment with parathyroid hormone. The results of these studies support the hypothesis that PTH regulation of acid production in osteoclasts involves both adenylate cyclase and PKC as effectors. J. Cell. Biochem. 65:565–573. © 1997 Wiley-Liss Inc.     

The ion channel TRPM7 regulates zinc-depletion-induced MDMX degradation

Zinc deficiency has been linked to human diseases, including cancer. MDMX, a crucial zinc-containing negative regulator of p53, has been found to be amplified or overexpressed in various cancers and implicated in the cancer initiation and progression. We report here that zinc depletion by the ion chelator TPEN or Chelex resin results in MDMX protein degradation in a ubiquitination-independent and 20S proteasome-dependent manner. Restoration of zinc led to recovery of cellular levels of MDMX. Further, TPEN treatment inhibits growth of the MCF-7 breast cancer cell line, which is partially rescued by overexpression of MDMX. Moreover, in a mass-spectrometry-based proteomics analysis, we identified TRPM7, a zinc-permeable ion channel, as a novel MDMX-interacting protein. TRPM7 stabilizes and induces the appearance of faster migrating species of MDMX on SDS-PAGE. Depletion of TRPM7 attenuates, while TRPM7 overexpression facilitates, the recovery of MDMX levels upon adding back zinc to TPEN-treated cells. Importantly, we found that TRPM7 inhibition, like TPEN treatment, decreases breast cancer cell MCF-7 proliferation and migration. The inhibitory effect on cell migration upon TRPM7 inhibition is also partially rescued by overexpression of MDMX. Together, our data indicate that TRPM7 regulates cellular levels of MDMX in part by modulating the intracellular Zn²⁺ concentration to promote tumorigenesis.     

Synthesis and antifungal activity of two novel spermidine analogues

Two spermidine analogues were synthesised and examined for antifungal activity. Both compounds used as 1 mM post-inoculation sprays reduced infection of barley seedlings by the powdery mildew fungus, Erysiphe graminis f.sp. hordei, infection of broad bean seedlings by the rust fungus, Uromyces viciae-fabae, and infection of apple seedlings by the powdery mildew fungus, Podosphaera leucotricha. Since these fungal pathogens cannot be cultured axenically, the effects of the two spermidine analogues on mycelial growth in vitro, as well as preliminary investigations on polyamine biosynthesis, were undertaken using the oat stripe pathogen, Pyrenophora avenae. Although neither compound affected radial growth of the fungus on plates, both analogues reduced fungal biomass in liquid culture substantially. The two spermidine analogues, used at a concentration of 1 mM, had no significant effect on the conversion of labelled ornithine into polyamines in P. avenae.     

Can puzzle feeders be used as cognitive screening instruments? Differential performance of young and aged female monkeys on a puzzle feeder task

Conventional cognitive testing of monkeys is time‐consuming and involves single‐caging and food or water deprivation. Here we report a novel test of global cognitive performance that can be completed in a short time period without food/water or social restrictions. Nine mazes of increasing difficulty were developed using a standard puzzle feeder, and the maze‐solving performance of ten young and five aged female cynomolgus monkeys (Macaca fascicularis) was tested. The young monkeys solved maze configurations at higher levels of difficulty and solved the first level of difficulty more quickly than aged monkeys. This task discriminated performance by age in nonhuman primates as do more conventional forms of cognitive testing and indicates that this task may be a quick and easy assessment of global cognitive function. Am. J. Primatol. 49:195–202, 1999. © 1999 Wiley‐Liss, Inc.     

Risk Factors and Outcomes of Invasive Fungal Infections in Allogeneic Hematopoietic Cell Transplant Recipients

Allogeneic hematopoietic cell transplant (HCT) recipients are at increased risk of invasive fungal infections (IFI), which are associated with a high mortality rate. We evaluated the impact of IFI in allogeneic HCT patients. In total, 541 consecutive allogeneic HCT recipients were included. The cumulative incidence of any IFI and mold infections at 1-year post-HCT was 10 and 7%, respectively. Median times to IFI and mold infection were 200 and 210 days, respectively. There was a trend toward fewer IFI and mold infections in the last several years. Both acute graft-versus-host disease (GVHD) (OR 1.83, p = 0.05) and corticosteroid duration (OR 1.0, p = 0.026) were significantly associated with increased risk of IFI, acute GVHD (OR 2.3, p = 0.027) emerged as the most important association with mold infections. Any IFI [HR 4.1 (2.79–6.07), p < 0.0001] and mold infections [HR 3.34 (2.1–5.1), p < 0.0001] were independently associated with non-relapse mortality (NRM). This association persisted in the setting of both acute and chronic GVHD. Corticosteroid treatment for >90 days was also significantly associated with higher NRM [HR 1.9 (1.3–2.6), p < 0.0001]. This study highlights the impact of IFI on NRM among HCT patients. The decrease in number of IFI and mold infections over the last several years may reflect the benefit of prophylaxis with mold-active antifungal agents.