Embryonic expression of an Nkx2‐5/Cre gene using ROSA26 reporter mice

Summary: Nkx2‐5, one of the earliest cardiac‐specific markers in vertebrate embryos, was used as a genetic locus to knock in the Cre recombinase gene by homologous recombination. Offspring resulting from heterozygous Nkx2‐5/Cre mice mated to ROSA26 (R26R) reporter mice provided a model system for following Nkx2‐5 gene activity by β‐galactosidase (β‐gal) activity. β‐gal activity was initially observed in the early cardiac crescent, cardiomyocytes of the looping heart tube, and in the epithelium of the first pharyngeal arch. In later stage embryos (10.5–13.5 days postcoitum, dpc), β‐gal activity was observed in the stomach and spleen, the dorsum of the tongue, and in the condensing primordium of the tooth. The Nkx2‐5/Cre mouse model should provide a useful genetic resource to elucidate the role of loxP manipulated genetic targets in cardiogenesis and other developmental processes. genesis 31:176–180, 2001. © 2001 Wiley‐Liss, Inc.     

Effect of intracerebroventricular alpha-MSH on food intake, adiposity, c-Fos induction, and neuropeptide expression

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a hypothalamic neuropeptide proposed to play a key role in energy homeostasis. To investigate the behavioral, metabolic, and hypothalamic responses to chronic central alpha-MSH administration, alpha-MSH was infused continuously into the third cerebral ventricle of rats for 6 days. Chronic alpha-MSH infusion reduced cumulative food intake by 10.7% (P < 0.05 vs. saline) and body weight by 4.3% (P < 0.01 vs. saline), which in turn lowered plasma insulin levels by 29.3% (P < 0.05 vs. saline). However, alpha-MSH did not cause adipose-specific wasting nor did it alter hypothalamic neuropeptide mRNA levels. Central alpha-MSH infusion acutely activated neurons in forebrain areas such as the hypothalamic paraventricular nucleus, as measured by a 254% increase in c-Fos-like immunoreactivity (P < 0.01 vs. saline), as well as satiety pathways in the hindbrain. Our findings suggest that, although an increase of central melanocortin receptor signaling acutely reduces food intake and body weight, its anorectic potency wanes during chronic infusion and causes only a modest decrease of body weight.     

Effect of nonylphenol on serum testosterone levels and testicular steroidogenic enzyme activity in neonatal, pubertal, and adult rats.

Previous dose range-finding studies with nonylphenol (NP) administered to rats in a soy- and alfalfa-free diet showed apparent feminization of several endpoints in male rats at doses of 25 ppm and above. One possible mechanism contributing to these effects is a reduction of testosterone at critical developmental periods. The present study was conducted as an adjunct to a multigeneration study and was designed to examine the effect of NP on testosterone production. Male rats in the F1 and F2 generations were exposed through their dams or directly to various dietary doses of NP (0, 25, 200 and 750 ppm) throughout gestation and until sacrifice at either postnatal day 2 (PND2), PND50, or PND140. Male pups in the F3 generation were examined only on PND2. At PND2, serum testosterone levels were significantly decreased in all groups exposed to NP in the F1 generation, but not in the F2 or F3 generations. The activity of 17alpha-hydroxylase/C17, 20 lyase (P450c17) in PND2 testicular homogenates was not affected by NP treatment. In F1 and F2 PND50 and PND140 rats, NP treatment did not affect serum testosterone levels. The absolute dorsolateral prostate weight was increased in the 200 and 750 ppm dose groups only in the F1 PND50 rats, however, no significant effects were observed in other male reproductive organs. NP treatment did not affect P450c17 activity in microsomes prepared from testes of F1 PND50 or PND140 rats. However, P450c17 activity was significantly decreased in testicular microsomes of F(2) PND50 (200 and 750 ppm dose groups) and PND140 (25, 200, and 750 ppm dose groups) rats. A decrease in testicular beta-nicotinamide adenine dinucleotide phosphate (NADPH) P450 reductase was also observed in all PND50 and PND140 NP-exposed rats of the F1 and F2 generations. The ability of NP to directly inhibit P450c17 activity in vitro at concentrations of 1-100 microM was also demonstrated. These results indicate that NP can inhibit the activity of enzymes involved in testosterone synthesis, but suggest minimal effects on testosterone or testosterone-dependent endpoints via this mechanism.     

Herpes Simplex Virus Products in Productive and Abortive Infection: II. Electron Microscopic and Immunological Evidence for Failure of Virus Envelopment as a Cause of Abortive Infection

Herpes simplex virus strain MPdk(-) multiplies in HEp-2 cells, but not in dog kidney (DK) cells. Strain MPdk(+)sp, a multistep mutant of MPdk(-), multiplies in both HEp-2 and DK cells. Stabilized lysates of productively infected cells yield three macromolecular aggregates of viral deoxyribonucleic acid and protein banding in CsCl gradients at densities of 1.285 g/cm(3) (alpha), 1.325 g/cm(3) (beta), and 1.37 to 1.45 g/cm(3) (gamma). Similar lysates from abortively infected cells yield only the beta and gamma bands. Electron microscopic examination revealed that (i) the alpha band contained enveloped nucleocapsids, whereas the beta band contained naked nucleocapsids and particles tentatively identified as internal components of the nucleocapsids, and that (ii) the enveloped virions and reduplication of cellular membranes observed in thin sections of productively infected cells were absent from abortively infected cells. Studies of the surface antigens of infected cells in a cytolytic system described previously revealed that abortively infected cells contained approximately 10-fold less virus-induced surface antigen than did productively infected cells. From these and other data published previously, we concluded that infectious MPdk(-) virions are not made in DK cells because (i) functional viral products necessary for the envelopment of the nucleocapsid are not made, and (ii) capsid proteins and some nonstructural products specified by the virus malfunction.     

The secrets of lemur teeth

New tools are available for teasing out aspects of life‐history variation among extinct species. Here we summarize research on the life histories of the extinct lemurs of Madagascar. There is a wide range of variation in dental developmental timing among these species, from among the most accelerated (Palaeopropithecus) to among the most prolonged (Hadropithecus) within the Order Primates. Rather than reflecting variation in body size, this diversity appears to relate to niche characteristics and encephalization.     

Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease

Intracranial transplantation of neural stem cells (NSCs) delayed disease onset, preserved motor function, reduced pathology and prolonged survival in a mouse model of Sandhoff disease, a lethal gangliosidosis. Although donor-derived neurons were electrophysiologically active within chimeric regions, the small degree of neuronal replacement alone could not account for the improvement. NSCs also increased brain beta-hexosaminidase levels, reduced ganglioside storage and diminished activated microgliosis. Additionally, when oral glycosphingolipid biosynthesis inhibitors (beta-hexosaminidase substrate inhibitors) were combined with NSC transplantation, substantial synergy resulted. Efficacy extended to human NSCs, both to those isolated directly from the central nervous system (CNS) and to those derived secondarily from embryonic stem cells. Appreciating that NSCs exhibit a broad repertoire of potentially therapeutic actions, of which neuronal replacement is but one, may help in formulating rational multimodal strategies for the treatment of neurodegenerative diseases.     

Quantitative assessment of hemoglobin-induced endothelial barrier dysfunction.

Hemoglobin (Hb)-based O2 carriers (HBOC) are undergoing extensive development as potential "blood substitutes." A major problem associated with these molecules is an increase in microvascular permeability and peripheral vascular resistance. In this paper, we utilized bovine lung microvascular endothelial cell monolayers and simultaneously measured Hb-induced changes in transendothelial electrical resistance, diffusive albumin permeability, and diffusive Hb permeability (PDH) for three forms of Hb: natural tetrameric human Hb-A and two polymerized recombinant HBOCs containing alpha-human and beta-bovine chains designated Hb-Polytaur (molecular mass: 500 kDa) and Hb-(Polytaur)n (molecular mass: approximately 1,000,000 Da), respectively. Hb-Polytaur and Hb-(Polytaur)n are being evaluated for clinical use as HBOCs. All three Hb molecules induce a rapid decline of transendothelial electrical resistance to 30% of baseline. Diffusive albumin permeabiltiy increases, on average, approximately ninefold (2.78 x 10(-7) vs. 2.47 x 10(-6) cm/s) in response to Hb exposure. All three Hb molecules induce an increase in their own permeability, a process that we have called Hb-induced Hb permeability. The magnitude of change of PDH is also related to Hb size. When PDH is corrected for the diffusive coefficient for each Hb species, no evidence of restricted diffusion is found. Immunofluorescent images demonstrate Hb-induced actin stress fiber formation and large intercellular gaps. These data provide the first quantitative assessment of the effect of polymerized HBOC on their own diffusion rates over time. We discuss the importance of these findings in terms of Hb extravasation rates, molecular sieving, and clinical consequences of HBOC use.     

Taxonomic patterns in the nitrogen assimilation of soil prokaryotes

Nitrogen (N) is frequently a limiting nutrient in soil; its availability can govern ecosystem functions such as primary production and decomposition. Assimilation of N by microorganisms impacts the availability of N in soil. Despite its established ecological significance, the contributions of microbial taxa to N assimilation are unknown. Here we measure N uptake and use by microbial phylotypes and taxonomic groups within a diverse assemblage of soil microbes through quantitative stable isotope probing (qSIP) with ¹⁵N. Following incubation with ¹⁵ , distinct patterns of ¹⁵N assimilation among taxonomic groups were observed. For instance, glucose addition stimulated ¹⁵N assimilation in most members of Actinobacteria and Proteobacteria but generally decreased ¹⁵N use by Firmicutes and Bacteriodetes. While is considered a preferred and universal source of N to prokaryotes, the majority (> 80%) of N assimilation in our soils could be attributed to a handful of active orders. Characterizing N assimilation of taxonomic groups with ¹⁵N qSIP may provide a basis for understanding how microbial community composition influences N availability in the environment.