Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition into the subendothelial basement membrane

Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. Biosynthetically radiolabeled proteins secreted into the culture medium were analyzed by sodium dodecyl sulfate gel electrophoresis after reduction, revealing a single collagenous component with an approximate Mr of 180 000 that could be resolved into two closely migrating polypeptide chains. Prior to reduction, the 180 000 bands migrated as a high molecular weight complex, indicating the presence of intermolecular disulfide bonding. The 180 000 material was identified as type IV procollagen on the basis of its selective degradation by purified bacterial collagenase, moderate sensitivity to pepsin digestion, immunoprecipitation with antibodies to human type IV collagen, and comigration with type IV procollagen purified from human and murine sources. In the basement membrane like matrix elaborated by the microvascular endothelial cells at their basal surface, type IV procollagen was the predominant constituent. This matrix-associated type IV procollagen was present as a highly cross-linked and insoluble complex that was solubilized only after denaturation and reduction of disulfide bonds. In addition, there was evidence of nonreducible dimers and higher molecular weight aggregates of type IV procollagen. These findings support the suggestion that the presence of intermolecular disulfide bonds and other covalent interactions stabilizes the incorporation of the type IV procollagen into the basement membrane matrix. Cultured microvascular endothelial cells therefore appear to deposit a basal lamina-like structure that is biochemically similar to that formed in vivo, providing a unique model system that should be useful for understanding microvascular basement membrane metabolism, especially as it relates to wound healing, tissue remodeling, and disease processes.     

Computational assessment of folding energy landscapes in heterodimeric coiled coils

The coiled coil structural motif consists of alpha helices supercoiling around each other to form staggered knobs‐into‐holes packing. Such structures are deceptively simple, especially as they often can be described with parametric equations, but are known to exist in various conformations. Even the simplest systems, consisting of 2 monomers, can assemble into a wide range of states. They can form canonical as well as noncanonical coiled coils, be parallel or antiparallel, where helices associate with different degrees of shift, tilt, and rotation. Here, we investigate the energy landscape of heterodimeric coiled coils by carrying out de novo folding simulations starting from amino acid sequence. We folded a diverse set of 22 heterodimers and demonstrate that the approach is capable of identifying the atomic details in the experimental structure in the majority of cases. Our methodology also enables exploration of alternative states that can be accessible in solution beyond the experimentally determined structure. For many systems, we observe folding energy landscapes with multiple energy minima and several isoenergetic states. By comparing coiled coils from single domains and those extracted from larger proteins, we find that standalone coiled coils have deeper energy wells at the experimentally determined conformation. By folding the competing homodimeric states in addition to the heterodimers, we observe that the structural specificity towards the heteromeric state is often small. Taken together, our results demonstrate that de novo folding simulations can be a powerful tool to characterize structural specificity of coiled coils when coupled to assessment of energy landscapes.     

A method for the isolation and serial propagation of keratinocytes,endothelial cells,and fibroblasts from a single punch biopsy of human skin

Summary When multiple types of cells from normal and diseased human skin are required, techniques to isolate cells from small skin biopsies would facilitate experimental studies. The purpose of this investigation was to develop a method for the isolation and propagation of three major cell types (keratinocytes, microvascular endothelial cells, and fibroblasts) from a 4-mm punch biopsy of human skin. To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from the dermis by treatment with dispase. Keratinocytes were dissociated from the epidermis by trypsin and plated on a collagen-coated tissue culture petri dish. A combination of two commercial media (Serum-Free Medium and Medium 154) provided optimal growth conditions. To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated tissue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishment and growth of these cells. Epithelioid endothelial cells were separated from spindle-shaped endothelial cells and from dendritic cells by selective attachment toUlex europeus agglutinin I-coated paramagnetic beads. To establish fibroblasts, dermal explants depleted of keratinocytes and endothelial cells were attached to plastic by centrifugation, and fibroblasts were obtained by explant culture and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS). Using these isolation methods and growth conditions, two confluent T-75 flasks of keratinocytes, one confluent T-25 flask of purified endothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin. This method should prove useful in studies of human skin where three cell types must be grown in sufficient quantities for molecular and biochemical analysis.     

Cardiopulmonary control in sleeping Sprague-Dawley rats treated with hydralazine

Carley, David W., Sinisa M. Trbovic, Alex Bozanich, andMiodrag Radulovacki. Cardiopulmonary control in sleepingSprague-Dawley rats treated with hydralazine. J. Appl.Physiol. 83(6): 1954-1961, 1997.To test thehypothesis that hydralazine can suppress spontaneous sleep-relatedcentral apnea, respiratory pattern, blood pressure, and heart periodwere monitored in Sprague-Dawley rats. In random order and on separatedays, rats were recorded after intraperitoneal injection of1) saline or2) 2 mg/kg hydralazine. Normalizedminute ventilation(NI)declined significantly with transitions from wake tonon-rapid-eye-movement (NREM) sleep (5.1%;P = 0.01) and rapid-eye-movement (REM)sleep (4.2%; P = 0.022).Hydralazine stimulated respiration(NIincreased by 21%; P < 0.03) andeliminated the effect of state onNI. Bloodpressure decreased by 17% after hydralazine, and the correlationbetween fluctuations in mean blood pressure andNI changedfrom strongly positive during control recordings to weakly negativeafter hydralazine (P < 0.0001 foreach). Postsigh and spontaneous apneas were reduced during NREM and REMsleep after hydralazine (P < 0.05 for each). This suppression was strongly correlated with the reductionin blood pressure and with the degree of respiratory stimulation. Weconclude that mild hydralazine-induced hypotension leads to respiratory stimulation and apnea suppression.

     

Inclusion bodies in pinealocytes of the cotton rat (Sigmodon hispidus)

Pinealocytes of the cotton rat (Sigmodon hispidus) often contain large (2-6 micron diameter) intracytoplasmic inclusions, the function of which is not known. These inclusions may represent nucleolus-like bodies, mineral deposits, secretory products or viral inclusions. In this study these inclusions were classified as type A, B or C inclusions based on the amount of electron-dense material interspersed within the finely granular material comprising the bulk of these inclusions. Each type of inclusion was analyzed by X-ray microanalysis and enzymatic proteinaceous digestion. X-ray microanalysis of these inclusions differed both quantitatively and semiquantitatively from that of human or gerbil pineal concretions, the latter two of which are extracellular deposits. Pronase, a proteolytic enzyme, digested the electron-dense material only after longer times of tissue exposure to this enzyme in contrast to the easily digested, finely granular matrix-like material of these inclusions. Such intrapinealocytic inclusions have only been observed in the cotton rat. Their functional significance remains unknown.     

Precise annual changes in the numbers of "synaptic" ribbons and spherules in the rat pineal gland

Although pineal "synaptic" ribbons (SR) are frequently examined by means of quantitative electron microscopy, their functional significance remains unclear. The same is true for related structures--"synaptic" spherules (SPH). In the course of such studies, it has been noted that SR counts may differ from laboratory to laboratory. Because seasonal changes may play a role, a 2-year study was performed on male rats kept under routine laboratory conditions and killed at monthly intervals during daytime or nighttime. Both structures examined showed distinct day-night differences throughout the year, with higher numbers at night than during the day. There were significant annual changes in both SR and SPH during both daytime and nighttime. The comparison of the curves from the 2 years showed that they were virtually identical both during daytime and nighttime. The numbers of SR were the highest in October and the lowest in April; the numbers of SPH had two plateaus, one with lower values from November to April, and the other with higher values from May to October. It appears from the present study that SR and SPH numbers in the rat pineal gland show statistically significant and precisely timed seasonal changes that may well account for the variations of SR numbers in the different publications.