Replication of wild-type and mutant human cytomegalovirus in life-extended human diploid fibroblasts

A cDNA encoding the catalytic subunit of human telomerase was used to generate life-extended derivatives of primary human diploid fibroblasts. The life-extended cells supported efficient human cytomegalovirus (HCMV) replication. A subclone of the life-extended cells was generated containing the HCMV UL82 gene and used to isolate and propagate a virus that exhibited a profound growth defect after infection at a low input multiplicity.     

Genetic control of susceptibility to mite-associated ulcerative dermatitis

Variable susceptibility to mite-associated ulcerative dermatitis (MAUD) attributable to Myobia musculi infestation was studied in breeding stocks of sixteen inbred strains of mice. Statistical evaluation of 41 occurrences of lesion among 1,517 mice of the various strains indicated that significant differences in the frequency of MAUD were associated with differences in genetic background and in the H-2 type. These findings suggest that the occurrence of an ulcerative skin lesion in inbred mice exposed to M. musculi is controlled by at least two genes. Lesion susceptibility appears to be profoundly affected by a non-H-2 linked gene or gene combination shared by all C57Bl background strains. In addition, a major influence is exerted by one or more genes within the H-2 complex.     

Metamorphosis alters the response to spinal cord transection in Xenopus laevis frogs

A series of studies has examined the response of the spinal cord to lesions made at various stages prior to and after metamorphic climax in the clawed frog Xenopus laevis. Complete transections made between Nieuwkoop and Faber (1956) stages 50 and 62 were followed by gradual recovery of righting and coordinated swimming as animals metamorphosed into juveniles (stage 66). Examination of descending axonal projections using horseradish peroxidase (HRP) showed fibers crossing the lesion site and distributing to the caudal lumbar spinal cord. These fibers could be traced from more rostral spinal segments as well as from brainstem injections of HRP. No evidence for rostrally projecting fibers crossing the lesion was obtained. Juvenile frogs of varying ages failed to demonstrate recovery of coordinated swimming or reconstitution of spinal descending pathways. In an additional series of animals, spinal transections were made within 1 or 2 days of tail resorption to assess whether regenerative capacities extended at all into post-metamorphic stages. No evidence for regeneration was found. Studies of metamorphosing frogs after spinal transections showed that fibers crossed the lesion within 5-12 days of transection, well prior to the end of metamorphic climax; however, in some cases in which metamorphosis seemed arrested, little regeneration was observed. Immunocytochemical studies showed that fibers containing serotonin (5-HT) were included in the population of axons that rapidly crossed the lesion after transection at metamorphic stages. These results are compared to those for lesions of the dorsal columns and other systems in developing and juvenile Xenopus. It is suggested that both metamorphosis-related hormonal changes, and axon substrate pathways, may affect the regenerative response in the Xenopus central nervous system (CNS).     

Novel technique for monitoring micturition and sexual function in male rats using telemetry

We developed a novel technique to simultaneously monitor micturitions and erections in rats by using pressure monitoring within the corpus spongiosum of the penis (CSP). We present data validating this technique and report pressure waveform characteristics of micturition and erectile events during four different behavioral contexts in 10 awake, freely-moving male rats. Telemetric pressure transducers were implanted in the bulb of the CSP. CSP pressure was monitored while the animals were simultaneously recorded on video for determination of presence and volume (n = 7) of micturitions and while the animals underwent behavioral tests for determination of erections. Observed micturitions and CSP pressure waveforms characteristic of micturitions occurred simultaneously (r = 0.98) at a frequency of 32 +/- 4 micturitions per 24 h and with a volume of 0.95 +/- 0.12 ml/urination. Micturition duration recorded by CSP pressure and volume determined by urine weight were highly correlated (r = 0.82). We found that 100% of visually confirmed erectile events occurred simultaneously with CSP pressure waveforms characteristic of erections during ex copula reflex erection tests. During noncontact erection and mating tests more erections were identified by telemetry than by observation alone. Erections during mating tests had a different appearance than those seen in other contexts; they were shorter in duration (P < 0.05) and typically were characterized by a single suprasystolic CSP pressure peak, highlighting the context-specificity of erections. Quality of recordings remained stable in three of four rats we followed for 8 wk. We demonstrate that telemetric recording of CSP pressure provides a quantitative and qualitative assessment of penile erections and micturition in freely behaving rats.     

Perinatal stem cells: A promising cell resource for tissue engineering of craniofacial bone

In facing the mounting clinical challenge and suboptimal techniques of craniofacial bone defects resulting from various conditions, such as congenital malformations, osteomyelitis, trauma and tumor resection, the ongoing research of regenerative medicine using stem cells and concurrent advancement in biotechnology have shifted the focus from surgical reconstruction to a novel stem cell-based tissue engineering strategy for customized and functional craniofacial bone regeneration. Given the unique ontogenetical and cell biological properties of perinatal stem cells, emerging evidence has suggested these extraembryonic tissue-derived stem cells to be a promising cell source for extensive use in regenerative medicine and tissue engineering. In this review, we summarize the current achievements and obstacles in stem cell-based craniofacial bone regeneration and subsequently we address the characteristics of various types of perinatal stem cells and their novel application in tissue engineering of craniofacial bone. We propose the promising feasibility and scope of perinatal stem cell-based craniofacial bone tissue engineering for future clinical application.     

Evolution of biological interaction networks: from models to real data

We are beginning to uncover common mechanisms leading to the evolution of biological networks. The driving force behind these advances is the increasing availability of comparative data in several species.     

BH3‐in‐groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes

Pro‐apoptotic Bax induces mitochondrial outer membrane permeabilization (MOMP) by forming oligomers through a largely undefined process. Using site‐specific disulfide crosslinking, compartment‐specific chemical labeling, and mutational analysis, we found that activated integral membrane Bax proteins form a BH3‐in‐groove dimer interface on the MOM surface similar to that observed in crystals. However, after the α5 helix was released into the MOM, the remaining interface with α2, α3, and α4 helices was rearranged. Another dimer interface was formed inside the MOM by two intersected or parallel α9 helices. Combinations of these interfaces generated oligomers in the MOM. Oligomerization was initiated by BH3‐in‐groove dimerization, without which neither the other dimerizations nor MOMP occurred. In contrast, α9 dimerization occurred downstream and was required for release of large but not small proteins from mitochondria. Moreover, the release of large proteins was facilitated by α9 insertion into the MOM and localization to the pore rim. Therefore, the BH3‐in‐groove dimerization on the MOM nucleates the assembly of an oligomeric Bax pore that is enlarged by α9 dimerization at the rim.     

Identification of binary interactions between human cytomegalovirus virion proteins

Human cytomegalovirus (HCMV) virions are composed of a DNA-containing nucleocapsid surrounded by a tegument layer and host-derived lipid envelope studded with virally encoded glycoproteins. These complex virions are estimated to be composed of more than 50 viral proteins. Assembly of HCMV virions is poorly understood, especially with respect to acquisition of the tegument; however, it is thought to involve the stepwise addition of virion components through protein-protein interactions. We sought to identify interactions among HCMV virion proteins using yeast two-hybrid analysis. Using 33 known capsid and tegument proteins, we tested 1,089 pairwise combinations for binary interaction in the two-hybrid assay. We identified 24 interactions among HCMV virion proteins, including 13 novel interactions among tegument proteins and one novel interaction between capsid proteins. Several of these novel interactions were confirmed by coimmunoprecipitation of protein complexes from transfected cells. In addition, we demonstrate three of these interactions in the context of HCMV infection. This study reveals several new protein-protein interactions among HCMV tegument proteins, some of which are likely important for HCMV replication and pathogenesis.