Increased mRNA levels for components of the lysosomal, Ca2+-activated, and ATP-ubiquitin-dependent proteolytic pathways in skeletal muscle from head trauma patients.

The cellular mechanisms responsible for enhanced muscle protein breakdown in hospitalized patients, which frequently results in lean body wasting, are unknown. To determine whether the lysosomal, Ca2+-activated, and ubiquitin-proteasome proteolytic pathways are activated, we measured mRNA levels for components of these processes in muscle biopsies from severe head trauma patients. These patients exhibited negative nitrogen balance and increased rates of whole-body protein breakdown (assessed by [13C]leucine infusion) and of myofibrillar protein breakdown (assessed by 3-methylhistidine urinary excretion). Increased muscle mRNA levels for cathepsin D, m-calpain, and critical components of the ubiquitin proteolytic pathway (i.e., ubiquitin, the 14-kDa ubiquitin-conjugating enzyme E2, and proteasome subunits) paralleled these metabolic adaptations. The data clearly support a role for multiple proteolytic processes in increased muscle proteolysis. The ubiquitin proteolytic pathway could be activated by altered glucocorticoid production and/or increased circulating levels of interleukin 1beta and interleukin 6 observed in head trauma patients and account for the breakdown of myofibrillar proteins, as was recently reported in animal studies.

Modulation of cognition-specific cortical activity by gonadal steroids: A positron-emission tomography study in women

There is considerable evidence from animal studies that gonadal steroid hormones modulate neuronal activity and affect behavior. To study this in humans directly, we used H215O positron-emission tomography to measure regional cerebral blood flow (rCBF) in young women during three pharmacologically controlled hormonal conditions spanning 4–5 months: ovarian suppression induced by the gonadotropin-releasing hormone agonist leuprolide acetate (Lupron), Lupron plus estradiol replacement, and Lupron plus progesterone replacement. Estradiol and progesterone were administered in a double-blind cross-over design. On each occasion positron-emission tomography scans were performed during (i) the Wisconsin Card Sorting Test, a neuropsychological test that physiologically activates prefrontal cortex (PFC) and an associated cortical network including inferior parietal lobule and posterior inferolateral temporal gyrus, and (ii) a no-delay matching-to-sample sensorimotor control task. During treatment with Lupron alone (i.e., with virtual absence of gonadal steroid hormones), there was marked attenuation of the typical Wisconsin Card Sorting Test activation pattern even though task performance did not change. Most strikingly, there was no rCBF increase in PFC. When either progesterone or estrogen was added to the Lupron regimen, there was normalization of the rCBF activation pattern with augmentation of the parietal and temporal foci and return of the dorsolateral PFC activation. These data directly demonstrate that the hormonal milieu modulates cognition-related neural activity in humans.

Cloning and characterization of TDD5, an androgen target gene that is differentially repressed by testosterone and dihydrotestosterone

By using mRNA polymerase chain reaction differential display technique (DDPCR), we have identified one early responsive cDNA fragment, TDD5, from a 5α-reductase-deficient T cell hybridoma. The DDPCR profiles of TDD5 suggest that its expression can be repressed by testosterone (T) within 2 hr. More importantly, both DDPCR and Northern blot analysis further demonstrated that the expression of TDD5 was differentially repressed by T and dihydrotestosterone (DHT) at the mRNA level. To our knowledge, this is the first androgen target gene to show a preference in response to T over DHT in cell culture. TDD5 is expressed in several tissues with particular abundance in kidney. Full-length TDD5 cDNA (2,916 bp) encodes a protein with a calculated molecular weight of 42,000. Finally, our animal studies further confirm that TDD5 mRNA levels can be repressed to the basal level 8 hr after DHT administration. The isolation and characterization of the early-responsive androgen target gene TDD5 and the fact that TDD5 mRNA level can be differentially regulated by T and DHT may provide a useful tool to study the molecular mechanism of androgen preference on target gene regulation.

Rhesus monkeys know when they remember

Humans are consciously aware of some memories and can make verbal reports about these memories. Other memories cannot be brought to consciousness, even though they influence behavior. This conspicuous difference in access to memories is central in taxonomies of human memory systems but has been difficult to document in animal studies, suggesting that some forms of memory may be unique to humans. Here I show that rhesus macaque monkeys can report the presence or absence of memory. Although it is probably impossible to document subjective, conscious properties of memory in nonverbal animals, this result objectively demonstrates an important functional parallel with human conscious memory. Animals able to discern the presence and absence of memory should improve accuracy if allowed to decline memory tests when they have forgotten, and should decline tests most frequently when memory is attenuated experimentally. One of two monkeys examined unequivocally met these criteria under all test conditions, whereas the second monkey met them in all but one case. Probe tests were used to rule out “cueing” by a wide variety of environmental and behavioral stimuli, leaving detection of the absence of memory per se as the most likely mechanism underlying the monkeys' abilities to selectively decline memory tests when they had forgotten.

The affective component of pain in rodents: Direct evidence for a contribution of the anterior cingulate cortex

Numerous human and animal studies indirectly implicate neurons in the anterior cingulate cortex (ACC) in the encoding of the affective consequences of nociceptor stimulation. No causal evidence, however, has been put forth linking the ACC specifically to this function. Using a rodent pain assay that combines the hind-paw formalin model with the place-conditioning paradigm, we measured a learned behavior that directly reflects the affective component of pain in the rat (formalin-induced conditioned place avoidance) concomitantly with “acute” formalin-induced nociceptive behaviors (paw lifting, licking, and flinching) that reflect the intensity and localization of the nociceptive stimulus. Destruction of neurons originating from the rostral, but not caudal, ACC reduced formalin-induced conditioned place avoidance without reducing acute pain-related behaviors. These results provide evidence indicating that neurons in the ACC are necessary for the “aversiveness” of nociceptor stimulation.

Ethinylestradiol does not enhance the expression of nitric oxide synthase in bovine endothelial cells but increases the release of bioactive nitric oxide by inhibiting superoxide anion production.

Estradiol is known to exert a protective effect against the development of atherosclerosis, but the mechanism by which this protection is mediated is unclear. Since animal studies strongly suggest that production of endothelium-derived relaxing factor is enhanced by estradiol, we have examined the effect of estrogens on nitric oxide (NO) synthase (NOS) activity, protein, and mRNA in cultured bovine aortic endothelial cells. In reporter cells rich in guanylate cyclase, it has been observed that long-term treatment (> or = 24 hr) with ethinylestradiol (EE2) dose-dependently increased guanylate cyclase-activating factor activity in the conditioned medium of endothelial cells. However, conversion of L-[14C]arginine to L-[14C]citrulline by endothelial cell homogenate or quantification of nitrite and nitrate released by intact cells in the conditioned medium did not reveal any change in NOS activity induced by EE2 treatment. Similarly, Western and Northern blot analyses did not reveal any change in the endothelial NOS protein and mRNA content in response to EE2. However, EE2 dose- and time-dependently decreased superoxide anion production in the conditioned medium of endothelial cells with an EC50 value (0.1 nM) close to that which increased guanylate cyclase-activating factor activity (0.5 nM). Both of these effects were completely prevented by the antiestrogens tamoxifen and RU54876. Thus, endothelium exposure to estrogens appears to induce a receptor-mediated antioxidant effect that enhances the biological activity of endothelium-derived NO. These effects could account at least in part for the vascular protective properties of these hormones.

Estrogen receptor α, not β, is a critical link in estradiol-mediated protection against brain injury

Estradiol protects against brain injury, neurodegeneration, and cognitive decline. Our previous work demonstrates that physiological levels of estradiol protect against stroke injury and that this protection may be mediated through receptor-dependent alterations of gene expression. In this report, we tested the hypothesis that estrogen receptors play a pivotal role in mediating neuroprotective actions of estradiol and dissected the potential biological roles of each estrogen receptor (ER) subtype, ERα and ERβ, in the injured brain. To investigate and delineate these mechanisms, we used ERα-knockout (ERαKO) and ERβ-knockout (ERβKO) mice in an animal model of stroke. We performed our studies by using a controlled endocrine paradigm, because endogenous levels of estradiol differ dramatically among ERαKO, ERβKO, and wild-type mice. We ovariectomized ERαKO, ERβKO, and the respective wild-type mice and implanted them with capsules filled with oil (vehicle) or a dose of 17β-estradiol that produces physiological hormone levels in serum. One week later, mice underwent ischemia. Our results demonstrate that deletion of ERα completely abolishes the protective actions of estradiol in all regions of the brain; whereas the ability of estradiol to protect against brain injury is totally preserved in the absence of ERβ. Thus, our results clearly establish that the ERα subtype is a critical mechanistic link in mediating the protective effects of physiological levels of estradiol in brain injury. Our discovery that ERα mediates protection of the brain carries far-reaching implications for the selective targeting of ERs in the treatment and prevention of neural dysfunction associated with normal aging or brain injury.