Muscle architectural properties indicate a primary role in support for the pelvic limb of three-toed sloths (Bradypus variegatus).

Tree sloths evolved below-branch locomotion making them one of few mammalian taxa beyond primates for which suspension is nearly obligatory. Suspension requires strong limb flexor muscles that provide both propulsion and braking/support, and available locomotor kinetics data indicate that these roles differ between fore- and hindlimb pairs. Muscle structure in the pelvic limb is hypothesized to be a key anatomical correlate of function in braking/support during suspensory walking and propulsion and/or support during vertical climbing. This expectation was tested by quantifying architecture properties in the hindlimb limb musculature of brown-throated three-toed sloths (Bradypus variegatus: N = 7) to distinguish the roles of the flexor/extensor functional muscle groups at each joint. Measurements of muscle moment arm (rm ), mass, belly length, fascicle length, pennation angle, and physiological cross-sectional area (PCSA) were taken from n = 45 muscles. Overall, most muscles studied show properties for contractile excursion and fast joint rotational velocity. However, the flexor musculature is more massive (p = 0.048) and has larger PCSA (p = 0.003) than the extensors, especially at the knee joint and digits where well-developed and strong flexors are capable of applying large joint torque. Moreover, selected hip flexors/extensors and knee flexors have modified long rm that can amplify applied joint torque in muscles with otherwise long, parallel fascicles, and one muscle (m. iliopsoas) was capable of moderately high power in B. variegatus. The architectural properties observed in the hip flexors and extensors match well with roles in suspensory braking and vertical propulsion, respectively, whereas strong knee flexors and digital flexors appear to be the main muscles providing suspensory support in the pelvic limb. With aid in support by the forelimbs and the use of adaptive slow locomotion and slow muscle fiber recruitment patterns, structure-function in the tensile limb systems of sloths appears to collectively represent an additional mechanism for energy conservation.

A designed Zn2+-binding amphiphilic polypeptide: energetic consequences of pi-helicity.

The pi-helix is a secondary structure with 4.4 amino acids per helical turn. Although it was proposed in 1952, no experimental support for its existence was obtained until the mid-1980s. While short peptides are unlikely to assume a marginally stable secondary structure spontaneously, they might do so in the presence of appropriate structural constraints. In this paper, we describe a peptide that is designed to assume a pi-helical conformation when stabilized by cetyltrimethylammonium bromide (CTAB) micelles and Zn(2+). In the designed peptide, lipophilic amino acids are placed such that it would be amphiphilic in the pi-helical, but not in the alpha-helical, conformation. Also, two His residues are incorporated with i, i + 5 spacing, designed to allow binding of Zn(2+) in a pi-helical but not an alpha-helical conformation. The peptide was found to form moderately stable monolayers at the air-water interface, with a collapse pressure that almost doubled when there was Zn(2+) in the subphase. Also, CTAB micelles induced a marked increase in the helicity of the peptide. In 50% TFE, the peptide had a CD spectrum consistent with an alpha-helical structure. The addition of 1 mM Zn(2+) to this solvent caused a saturable decline in ellipticity to approximately half of its original value. The peptide also bound Zn(2+) when it was bound to CTAB micelles, with Zn(2+) again inducing a decrease in ellipticity. The peptide had slightly greater affinity for Zn(2+) in the presence of the CTAB than in a 50% TFE solution (K(d) = 3.1 x 10(-4) M in CTAB and 2.3 x 10(-4) M in TFE). van't Hoff analysis indicated that thermal denaturation of the peptide in 50% TFE containing 1 mM Zn(2+) was associated with both enthalpic and entropic changes that were greater than those in the absence of Zn(2+). These observations are all consistent with the proposal that the peptide assumed a pi-helical conformation in the presence of Zn(2+) and CTAB micelles, and has allowed the stability of this rare conformation to be assessed.

Delayed testosterone replacement restores nitric oxide synthase-containing nerve fibres and the erectile response in rat penis.

OBJECTIVE: To elucidate the effect of testosterone on penile innervation. Materials and methods Three groups of six rats each were assessed; two groups (1 and 2) were castrated and the third (group 3) underwent a sham operation (control). Eight weeks after castration, group 2 received a subcutaneous injection with testosterone. At 8 weeks, the rats in group 1 and 3 underwent a final functional analysis while those in group 2 did so at 12 weeks. The evaluation included a subcutaneous injection with apomorphine to study centrally mediated erection, and cavernosal nerve electrostimulation and papaverine injection to study peripherally mediated erection. At death a penile mid-shaft specimen was taken for NADPH-diaphorase staining. RESULTS: In the apomorphine study, castration resulted in significantly fewer yawns and erections than in the control, and those in group 2 significantly better central erectile function than in the controls. The mean (SEM) number of nitric oxide synthase (NOS)-containing nerve fibres in the corpora cavernosa and both dorsal nerves of castrated rats, at 46.2 (9.1) and 203 (32.1), respectively, were significantly lower than in rats in group 2, at 84.1 (11.2) and 300.6 (17.1), and than in the controls, at 88.6 (10.9) and 306.3 (22.9), respectively. The intracavernosal pressure decreased significantly in the absence of testosterone, both after electrostimulation and intracavernosal papaverine injection. However, there was no difference between the control and group 2 rats in either the number of NOS-containing nerve fibres or in the peripheral erectile functional study. CONCLUSIONS: Testosterone acts on the nervous system to mediate erection; when it is absent there may be down-regulation of both the production and activity of NO, thereby decreasing the response to peripheral stimulation via the NO pathway. The restoration of erectile function seen in rats in group 2 supports this phenomenon. Delayed testosterone replacement has no detrimental effect on the restoration of the erectile mechanism after castration.

Polyamines. An overview.

The polyamines spermine, spermidine, and putrescine are small organic molecules one or more of which are present in all living organisms. Many natural products contain polyamine residues. Polyamines are synthesized by a highly regulated pathway from arginine or ornithine and also can be transported in and out of cells. Polyamines are degraded to a variety of compounds the functions of which are largely unknown. Polyamines influence the transcriptional and translational stages of protein synthesis, stabilize membranes, and, in mammalian systems, modulate neurophysiological functions and may act as intracellular messengers. However, at the molecular level the mode of action of the polyamines is largely unknown.

Differential regulation of gene transcription in subpopulations of human B lymphocytes by vitamin D3.

We have previously shown that freshly extirpated normal human tonsil B cells, which are phenotypically diverse, representing different stages of cellular activation and differentiation, are refractory to the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and require specific activation signals for induction of responsiveness. To determine whether these diversely activated B cell populations respond to 1,25-(OH)2D3, human tonsil B cells were density fractionated and evaluated biochemically and functionally. Low density tonsil B cells, representing the centroblastic fraction, were observed to constitutively express vitamin D receptor message and protein. In contrast, high density quiescent tonsillar B cells had no detectable vitamin D receptor message or protein and required stimulation in vitro for their up-regulation. Biological responsiveness to 1,25-(OH)2D3 was assessed by messenger RNA (mRNA) expression of the vitamin D-dependent enzyme, 25-hydroxyvitamin D3 24-hydroxylase. Low density centroblastic B cells did not require exogenous surface activation for expression of 24-hydroxylase mRNA, which was detectable after 6 h of culture in the presence of 1,25-(OH)2D3. In contrast, high density tonsil B cells required in vitro activation for induction of 24-hydroxylase mRNA, and expression was not detectable for up to 48 h of culture. These observations suggest that reactivity of normal B cell populations to vitamin D is dependent upon their specific stage of activation.

Effect of simulated birth trauma on the urinary continence mechanism in the rat.

OBJECTIVES: To examine the effect of simulated birth injury in an animal model as part of a study on the pathogenesis of stress urinary incontinence (SUI) and the urinary continence mechanism. METHODS: A balloon was inflated in the vaginas of rats for 4 hours to simulate prolonged labor. The effect on the continence mechanism was assessed by functional, anatomic, biochemical, and histologic examinations. The functional test consisted of placing chili powder or a clipped whisker into the rat's nostrils to induce sneezing. Anatomic measurement of the genital hiatus was performed with a caliper. Serum creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) were measured to examine the value of muscle injury in predicting incontinence. c-Fos immunostaining in the spinal cord was used as a marker of nerve injury. These data were then correlated with histopathologic examination of the urethra and pelvic floor tissues. RESULTS: Four weeks after simulated birth injury, SUI was noted in 19 of 48 experimental rats. The genital hiatus was significantly wider in incontinent rats. The serum CPK and LDH levels were markedly elevated, but no difference was noted between the continent and incontinent rats. All experimental rats showed many c-Fos immunostaining neurons in the L6 to S1 spinal cord segments, but none was seen in control rats. Histologic study revealed a marked decrease of ganglion cells in the neural plexuses posterolateral to the vagina in experimental rats. After 4 weeks, muscle necrosis and degeneration, irregular shape and size of muscle fibers, and a change in the type I/II ratio were prominent features in the levator ani. In the urethra, we noted a significant decline in urethral wall musculature (both smooth and striated) in incontinent rats. CONCLUSIONS: In this novel rat model, simulated birth injury resulted in SUI in a portion of the animals. Pathologic changes in the urethra, pelvic ganglia, and levator muscles seem to be the contributing factors to SUI.

Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells.

We have examined whether modulation of the polyamine biosynthetic pathway, through inhibition by alpha-difluoromethylornithine (DFMO) of the rate limiting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J774 macrophages. DFMO potentiated LPS-stimulated nitrite production in both a concentration- and time-dependent manner, increasing nitrite levels by 48+/-5% at 10 mM. This effect was observed in cells pre-treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS-induced nitrite production. Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS-induced nitrite production or in the ability of DFMO (10 mM) to potentiate LPS-induced NO synthesis. Metabolism of L-[3H]arginine to L-[3H]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by either DFMO (1-10 mM) or by putrescine (0.001-1 mM), spermidine (0.001-1 mM) or spermine (0.001-1 mM). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70+/-0.07%) by L-NMMA (100 microM). Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with LPS resulted in enhanced (approximately 2 fold) iNOS protein expression. These results show that DFMO potentiates LPS-induced nitrite production in the murine macrophage cell line J774. Since the only known mechanism of action of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we conclude that expression of iNOS can be critically regulated by endogenous polyamines.

Growth factors in bladder wound healing.

INTRODUCTION: Surgical and traumatic injuries to the bladder initiate a complex series of biological processes that result in wound healing. This process involves cellular proliferation, migration and differentiation; removal of damaged tissue; and production of extracellular matrix all of which may be controlled by growth factors. In skin, keratinocyte growth factor (KGF) is induced following incisional injury. We hypothesize that in bladder wound healing KGF and other growth factors are induced to modulate tissue repair. METHODS: We have created a model of surgical bladder injury in the rodent. At 12, 24 and 48 hrs and 5 and 7 days after injury, the bladder was bisected and total RNA extracted from the anterior or wounded half and posterior or non-wounded half. Histological analysis of the bladder wound was performed with Mason's Trichrome and immunohistochemistry against smooth muscle alpha actin. RNase protection assays were performed to examine the expression of KGF, transforming growth factor (TGF)alpha and TGF beta 2 and 3 as well as the receptors for KGF and epidermal growth factor (EGF). Lastly, the effects of the exogenous administration of KGF on the bladder was tested on neonatal mice by daily injections of 5 micrograms KGF per gram body weight for 5 days. RESULTS: At 12 hours after injury KGF mRNA expression in the anterior wounded bladder half and posterior non-wounded bladder half was 8 and 6 times higher respectively, compared to unoperated control bladders. A similar response was seen for TGF alpha, where the 12 hour mRNA expression was 4.5 times higher in the anterior wounded bladder half and 3.5 times higher in the posterior non-wounded bladder half compared to unoperated control bladders. The nadir mRNA expression for both KGF and TGF alpha occurred at 7 days after bladder injury and was the same as in unoperated control bladders. EGFR mRNA expression was approximately 2 times higher in both the anterior wounded and posterior non-wounded bladder halves compared to the nadir levels which occurred at 24 hours after injury. TGF beta 2 and beta 3 mRNA levels did not significantly change in either the anterior wounded or posterior non-wounded bladder halves. Exogenous KGF stimulation resulted in a marked urothelial proliferation when compared to age matched control animals. CONCLUSION: During the early phases of bladder wound healing (12-24 hours post injury), mRNA for KGF and TGF alpha increased, whereas TGF beta 2 and beta 3 and the KGFR and EGFR remain unchanged. Additionally, exogenous KGF has a direct effect on urothelial proliferation. KGF and TGF alpha warrant further study as potential mediators of bladder wound healing.

Age decreases nitric oxide synthase-containing nerve fibers in the rat penis.

PURPOSE: To study the effect of aging on erectile function in a rat model. MATERIALS AND METHODS: We investigated: 1) the number and distribution of nerve fibers within the corpus cavernosum and dorsal nerve containing vasoactive intestinal polypeptide (VIP) and nitric oxide synthase (NOS); and 2) the erectile response to apomorphine (a central dopamine receptor agonist), electrostimulation of the cavernous nerve, and intracorporeal papaverine injection. RESULTS: The number of NOS-containing nerve fibers was significantly less in the old rats (24 months) than in the young (2.5 months) and intermediate (8.5 months)-aged (63.3 +/- 3.35 vs. 135.1 +/- 10.88 [p < or = 0.0002] and 127.8 +/- 11.65 [p < or = 0.0002]). The number of erections induced by apomorphine was significantly less in the old rats than in the young (1.0 +/- 3.1 vs. 3.6 +/- 0.26; p < 0.002). With electrostimulation, the latency period before the onset of the intracavernous pressure rise was noted to increase with age (2.3 +/- 0.24 sec. for the young vs. 6.77 +/- 0.98 sec. for the old, p < or = 0.0001). The maximal intracavernous pressure after intracavernous papaverine injection decreased with age. CONCLUSION: The erectile mechanism appears to remain intact as rats age, but the response to central and peripheral stimulation decreases. The reduction in NOS-containing nerve fibers might account for these observations.

Requirements for induction of vitamin D-mediated gene regulation in normal human B lymphocytes.

Mature human lymphocytes are unique targets of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) in that vitamin D receptors (VDR) are not constitutively expressed, and specific cellular activation signals are required for both the up-regulation of VDR and establishment of reactivity to the lipophilic ligand. Treatment of B lymphocytes with the cytokine IL-4 (IL-4), in the absence of prior activation, induces a weak up-regulation of VDR expression but fails to generate vitamin D-responsive element (VDRE)-reactive nuclear protein complexes or to initiate the genomic transcription of 25-hydroxyvitamin D3 24-hydroxylase. Stimulation of B lymphocytes by either ligation of CD40 Ag or cross-linking the Ig receptor is also insufficient to render B lymphocytes responsive to 1 alpha,25(OH)2D3. However, this apparent lack of response to the secosterol can be overcome by stimulation of B lymphocytes with a combination of these cellular activation signals, which are sufficient to lead to G1 cell cycle progression. In the presence of 1 alpha,25(OH)2D3, cellular activation associated with stimulation of such a progression appears to be sufficient for the up-regulation of VDR message and protein and necessary for the establishment of VDRE binding complexes and the induction of 24-hydroxylase message. Furthermore, biologic functions are modulated, in that the hormone inhibits proliferation in a subset of the activated B cells. These observations suggest that reactivity to 1 alpha,25(OH)2D3 is tightly regulated in B lymphocytes, requiring specific signals for its initiation.