Faculty Bibliography

I entered the field of cell biology during its formative phase and contributed technical advances in electron microscopy that gave new insights into the physiological roles of subcellular compartments. I concentrated for many years on the structure and function of the rough endoplasmic reticulum where I aimed at explaining mechanistically how the functional specialization of ribosomes bound to the endoplasmic reticulum membranes is achieved. This work eventually led to the formulation of the signal hypothesis with Gunter Blobel in 1971. Subsequently, my laboratory contributed to the birth of the field of protein traffic with the demonstration that membrane-bound ribosomes in the ER are also responsible for the synthesis of membrane and luminal proteins destined to other subcellular compartments, pointing to the existence of sorting signals in the newly synthesized polypeptides and corresponding discriminating trafficking mechanisms. I have derived great satisfaction from the fact that some of the work I will present, including the introduction with M. Cerejido of the MDCK cell line to study the development and properties of polarized epithelia and the discovery with Rodriguez-Boulan of the polarized budding of enveloped viruses, has helped many laboratories to continue to explore the fascinating mechanisms that contribute to generate the complex organization of eukaryotic cells

Intranasal administration of neuropeptide Y (NPY) is a promising treatment strategy to reduce traumatic stress-induced neuropsychiatric symptoms of posttraumatic stress disorder (PTSD). We evaluated the potential of intranasal NPY to prevent dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis, a core neuroendocrine feature of PTSD. Rats were exposed to single prolonged stress (SPS), a PTSD animal model, and infused intranasally with vehicle or NPY immediately after SPS stressors. After 7 days undisturbed, hypothalamus and hippocampus, 2 structures regulating the HPA axis activity, were examined for changes in glucocorticoid receptor (GR) and CRH expression. Plasma ACTH and corticosterone, and hypothalamic CRH mRNA, were significantly higher in the vehicle but not NPY-treated group, compared with unstressed controls. Although total GR levels were not altered in hypothalamus, a significant decrease of GR phosphorylated on Ser232 and increased FK506-binding protein 5 mRNA were observed with the vehicle but not in animals infused with intranasal NPY. In contrast, in the ventral hippocampus, only vehicle-treated animals demonstrated elevated GR protein expression and increased GR phosphorylation on Ser232, specifically in the nuclear fraction. Additionally, SPS-induced increase of CRH mRNA in the ventral hippocampus was accompanied by apparent decrease of CRH peptide particularly in the CA3 subfield, both prevented by NPY. The results show that early intervention with intranasal NPY can prevent traumatic stress-triggered dysregulation of the HPA axis likely by restoring HPA axis proper negative feedback inhibition via GR. Thus, intranasal NPY has a potential as a noninvasive therapy to prevent negative effects of traumatic stress.

Disorders in skin wound healing are a major health problem that requires the development of innovative treatments. The use of biomaterials as an alternative of skin replacement has become relevant, but its use is still limited due to poor vascularization inside the scaffolds, resulting in insufficient oxygen and growth factors at the wound site. In this study, we have developed a cell-based wound therapy consisting of the application of collagen-based dermal scaffolds containing mesenchymal stem cells from Wharton's jelly (WJ-MSC) in an immunocompetent mouse model of angiogenesis. From our comparative study on the secretion profile between WJ-MSC and adipose tissue-derived MSC, we found a stronger expression of several well-characterized growth factors, such as VEGF-A, angiopoietin-1 and aFGF, which are directly linked to angiogenesis, in the culture supernatant of WJ-MSC, both on monolayer and 3D culture conditions. WJ-MSC proved to be angiogenic both in vitro and in vivo, through tubule formation and CAM assays, respectively. Moreover, WJ-MSC consistently improved the healing response in vivo in a mouse model of human-like dermal repair, by triggering angiogenesis and further providing a suitable matrix for wound repair, without altering the inflammatory response in the animals. Since these cells can be easily isolated, cultured with high expansion rates and cryopreserved, they represent an attractive stem cell source for their use in allogeneic cell transplant and tissue engineering.

Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the genera Neisseria and Moraxella have evolved two-component systems that can extract iron from the host glycoproteins lactoferrin and transferrin. The homologous iron-transport systems consist of a membrane-bound transporter and an accessory lipoprotein. While the mechanism behind iron acquisition from transferrin is well understood, relatively little is known regarding how iron is extracted from lactoferrin. Here, the crystal structure of the N-terminal domain (N-lobe) of the accessory lipoprotein lactoferrin-binding protein B (LbpB) from the pathogen Neisseria meningitidis is reported. The structure is highly homologous to the previously determined structures of the accessory lipoprotein transferrin-binding protein B (TbpB) and LbpB from the bovine pathogen Moraxella bovis. Docking the LbpB structure with lactoferrin reveals extensive binding interactions with the N1 subdomain of lactoferrin. The nature of the interaction precludes apolactoferrin from binding LbpB, ensuring the specificity of iron-loaded lactoferrin. The specificity of LbpB safeguards proper delivery of iron-bound lactoferrin to the transporter lactoferrin-binding protein A (LbpA). The structure also reveals a possible secondary role for LbpB in protecting the bacteria from host defences. Following proteolytic digestion of lactoferrin, a cationic peptide derived from the N-terminus is released. This peptide, called lactoferricin, exhibits potent antimicrobial effects. The docked model of LbpB with lactoferrin reveals that LbpB interacts extensively with the N-terminal lactoferricin region. This may provide a venue for preventing the production of the peptide by proteolysis, or directly sequestering the peptide, protecting the bacteria from the toxic effects of lactoferricin.

Three strains capable of rapidly degrading TBBPA by co-metabolism and utilizing formate as the carbon source, named as J-F-01, J-F-02, and J-F-03, respectively, were isolated from enrichment cultures, which have been treated with 0.5mg/L TBBPA for 240 d. Based on morphology and 16S rRNA gene sequence analysis, both J-F-01 and J-F-02 were determined to Pseudomonas sp., while J-F-03 was identified as Streptococcus sp. A shorter half-life (6.1d) of TBBPA was observed in pure culture of J-F-03 when compared with J-F-01 (22.5d) and J-F-02 (13.6d). Surprisingly, the degradation of TBBPA was significantly enhanced by the mixed culture of J-F-02 and J-F-03. The optimal degradation conditions for the mixed cultures were determined. Under the optimal conditions, TBBPA (0.5mg/L) was completely metabolized by the mixed culture within ten days. Moreover, bromide and the metabolisms were detected, and a possible metabolic pathway was deduced from the detection of metabolite production patterns.

Importance: Hermansky-Pudlak syndrome (HPS) is a rare genodermatosis characterized by oculocutaneous albinism, platelet dysfunction, and in some patients, pulmonary fibrosis and granulomatous colitis. The granulomatous inflammation in the bowel of patients with HPS can be indistinguishable clinically and histologically from that of Crohn disease (CD); however, mucocutaneous granulomatous lesions have not been considered among the typical skin findings of HPS. Observations: We report a case of an albino woman in her 40s with a history of CD and pulmonary fibrosis who presented with ulcers, plaques, and nodules in the vulva, perineum, inguinal creases, and left axilla. These cutaneous findings had the typical clinical and histologic findings of metastatic cutaneous CD. However, she also had a genetically confirmed diagnosis of HPS. Conclusions and Relevance: It is unclear whether our patient's cutaneous findings were due to CD or secondary to HPS. This report reviews the features of HPS and CD, 2 entities characterized by a granulomatous inflammatory reaction pattern but with unique genetic and clinical features, and discusses the possible overlap between the 2 diagnoses.

In recent decades, there has been growing interest in the field of gasotransmitters, endogenous gaseous signaling molecules (NO, H2S, and CO), as regulators of a multitude of biochemical pathways and physiological processes. Most of the concerted effort has been on eukaryotic gasotransmitters until the subsequent discovery of bacterial counterparts. While the fundamental aspects of bacterial gasotransmitters remain undefined and necessitate further research, we will discuss a known specific role they play in defense against antibiotics. Considering the current dilemma of multidrug-resistant bacteria we consider it particularly prudent to exploring novel targets and approaches, of which the bacterial gasotransmitters, nitric oxide and hydrogen sulfide represent.