Emerging photon technologies for probing ultrafast molecular dynamics.

The understanding of physical and chemical changes at an atomic spatial scale and on the time scale of atomic motion is essential for a broad range of scientific fields. A new class of femtosecond, intense, short wavelength lasers, the free electron lasers, has opened up new opportunities to investigate dynamics in many areas of science. For chemical dynamics to advance however, a rigorous, quantitative understanding of dynamical effects due to intense X-ray exposure is also required. We illustrate this point by reporting here an experimental and theoretical investigation of the interaction of C(60) molecules with intense X-ray pulses, in the multiphoton regime. We also describe the potential of new available instrumentation and explore their potential impact in physical, chemical and biological sciences when they are coupled with emerging photon technologies.

Femtosecond X-ray-induced explosion of C60 at extreme intensity.

Understanding molecular femtosecond dynamics under intense X-ray exposure is critical to progress in biomolecular imaging and matter under extreme conditions. Imaging viruses and proteins at an atomic spatial scale and on the time scale of atomic motion requires rigorous, quantitative understanding of dynamical effects of intense X-ray exposure. Here we present an experimental and theoretical study of C60 molecules interacting with intense X-ray pulses from a free-electron laser, revealing the influence of processes not previously reported. Our work illustrates the successful use of classical mechanics to describe all moving particles in C60, an approach that scales well to larger systems, for example, biomolecules. Comparisons of the model with experimental data on C60 ion fragmentation show excellent agreement under a variety of laser conditions. The results indicate that this modelling is applicable for X-ray interactions with any extended system, even at higher X-ray dose rates expected with future light sources.

Dynamics of hollow atom formation in intense x-ray pulses probed by partial covariance mapping.

When exposed to ultraintense x-radiation sources such as free electron lasers (FELs) the innermost electronic shell can efficiently be emptied, creating a transient hollow atom or molecule. Understanding the femtosecond dynamics of such systems is fundamental to achieving atomic resolution in flash diffraction imaging of noncrystallized complex biological samples. We demonstrate the capacity of a correlation method called "partial covariance mapping" to probe the electron dynamics of neon atoms exposed to intense 8 fs pulses of 1062 eV photons. A complete picture of ionization processes competing in hollow atom formation and decay is visualized with unprecedented ease and the map reveals hitherto unobserved nonlinear sequences of photoionization and Auger events. The technique is particularly well suited to the high counting rate inherent in FEL experiments.

Explosion of xenon clusters driven by intense femtosecond pulses of extreme ultraviolet light.

The explosions of large xenon clusters irradiated by intense, femtosecond extreme ultraviolet pulses at a wavelength of 38 nm have been studied. Using high harmonic generation from a 35 fs laser, clusters have been irradiated by extreme ultraviolet pulses at intensity approaching 10;{11} W/cm;{2}. Charge states up to Xe8+ are observed, states well above those produced by single atom illumination, indicating that plasma continuum lowering is important. Furthermore, the kinetic energy distribution of the exploding ions is consistent with a quasineutral hydrodynamic expansion, rather than a Coulomb explosion.

Variations in the complement regulatory genes factor H (CFH) and factor H related 5 (CFHR5) are associated with membranoproliferative glomerulonephritis type II (dense deposit disease).

INTRODUCTION: Membranoproliferative glomerulonephritis type II or dense deposit disease (MPGN II/DDD) causes chronic renal dysfunction that progresses to end stage renal disease in about half of patients within 10 years of diagnosis. Deficiency of and mutations in the complement factor H (CFH) gene are associated with the development of MPGN II/DDD, suggesting that dysregulation of the alternative pathway of the complement cascade is important in disease pathophysiology. SUBJECTS: Patients with MPGN II/DDD were studied to determine whether specific allele variants of CFH and CFHR5 segregate preferentially with the MPGN II/DDD disease phenotype. The control group was compromised of 131 people in whom age related macular degeneration had been excluded. RESULTS: Allele frequencies of four single nucleotide polymorphisms in CFH and three in CFHR5 were significantly different between MPGN II/DDD patients and controls. CONCLUSION: We have identified specific allele variants of CFH and CFHR5 associated with the MPGN II/DDD disease phenotype. While our data can be interpreted to further implicate complement in the pathogenesis of MPGN II/DDD, these associations could also be unrelated to disease pathophysiology. Functional studies are required to resolve this question.

Human factor H-related protein 5 (FHR-5). A new complement-associated protein.

A novel human plasma protein has been identified as a universal component of complement deposits, when complement is detected immunohistochemically in vivo. The protein is homologous to complement factor H and related proteins and has been designated factor H-related protein 5 (FHR-5). FHR-5 was identified by a monoclonal antibody raised using pathologic human glomerular preparations as the immunogen. FHR-5 was purified by affinity chromatography from complement-lysed erythrocytes, and the peptide sequence was obtained. The cDNA was cloned from a human liver library, and FHR-5 was deduced to be a protein containing 551 amino acids organized into nine short consensus repeat motifs. The short consensus repeats of FHR-5 show homology to Factor H and to other Factor H-related proteins, with some unique features demonstrated. Recombinant FHR-5, expressed in insect cells, was shown to bind C3b in vitro. The strong association of FHR-5 with tissue complement deposits in vivo suggests that this additional member of the Factor H family of proteins has a function in complement regulation.

Sublytic complement injury does not activate NF-kappa B, or induce mitogenesis in rat mesangial cells.

Sublytic complement injury to glomerular mesangial cells, mediated by the terminal membrane attack complex of complement (C5b-9), is a potential initiating mechanism in IgA nephropathy. Sublytic complement injury has been reported to result in the production of a variety of pro-inflammatory molecules and growth factors, including many regulated by the transcription factor NF-kappa B. To determine the importance of complement injury in the pro-inflammatory signalling which occurs in IgA nephropathy, we investigated NF-kappa B activation following sublytic complement injury to cultured rat glomerular mesangial cells (RMCs). A sublytic dose of rabbit anti-Thy 1.1 (THY) serum and normal human serum was selected based upon flow cytometry, chromium-release assay, and induction of superoxide production. No significant C5b-9-induced NF-kappa B activation was detected by electrophoretic mobility shift assays, luciferase activity of RMCs transfected with a NF-kappa B-driven luciferase reporter construct, nor by Northern blots for the NF-kappa B-responsive mRNA species monocyte chemoattractant protein-1 or I kappa B alpha. Furthermore, measurements of (3)H incorporation following sublytic complement injury showed inhibition of mesangial cell mitogenesis in comparison to the heat-inactivated serum treatment and to THY alone. The results of this study suggest that sublytic complement injury to RMC does not directly activate NF-kappa B nor induce mesangial cell proliferation in mesangial cells. Other mechanisms such as IgA immune complex formation must be required to produce these events in IgA nephropathy.

Localisation of clusterin in normal human sperm by immunogold electron microscopy.

In the human male reproductive tract, two forms of clusterin have been detected: the conventional heterodimeric form and a novel acrosomal form. On human sperm the novel form of clusterin is present in the acrosomal region of acrosome intact sperm only. The aim of this study was to determine the site of localisation of the acrosomal form of clusterin using immunogold electron microscopy on normal human sperm. Using the E5 anticlusterin mAb and a preembedding technique, acrosomal clusterin was localised in the acrosomal contents. Immunogold particles were detected on ethanol fixed spermatozoa that were subjected to Triton X-100 permeabilisation treatment. These sperm had lost their plasmalemma and outer acrosomal membrane. Specific immunogold labeling was present over the surface mainly of the acrosomal contents exposed by the loss of the plasma-lemma and outer acrosomal membrane. Immunogold particles were also detected in the equatorial segment of the sperm. These data confirm that the acrosomal form of clusterin is associated with the contents of the acrosome.

Cutaneous necrosis from calcific uremic arteriolopathy.

Calcific uremic arteriolopathy (calciphylaxis) is an uncommon complication of chronic renal failure that is associated with high morbidity and mortality. We report 16 patients (13 female) who presented between 1985 and 1996. All patients developed painful livido reticularis that progressed to cutaneous necrosis and ulceration (11 cases on the proximal extremities and five cases on the distal extremities). Two patients with predominately distal leg disease survived; the cause of death in the other 14 patients was sepsis (six patients), withdrawal from dialysis (three), cardiac arrest (three), and gastrointestinal hemorrhage (two). Mesenteric ischemia from intestinal vascular calcification occurred in two cases. Clinical factors identified included the use of warfarin therapy in seven cases and significant weight loss (>10% body weight) in seven cases in the 6 months preceding the development of calcific uremic arteriolopathy. Skin pathology was studied in 12 cases, with all showing calcific panniculitis and small vessel calcification. Electron microscopic spectral analysis of the mineral content of the calcific lesions in the subcutaneous tissue showed only calcium and phosphorous. In two cases, substitution of low molecular weight heparin for warfarin therapy resulted in clinical improvement. Current theories of pathogenesis and treatment are reviewed. This study confirms the high morbidity and mortality of calcific uremic arteriolopathy producing ischemic tissue necrosis while drawing attention to significant weight loss and warfarin therapy as risk factors for the development of ischemic tissue necrosis. Hyperbaric oxygen therapy warrants further study.

Heparin-binding epidermal growth factor-like growth factor, an immediate-early gene for mesangial cells, is up-regulated in the Thy-1.1 model.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is one of five well-described growth factors which bind to and activate the EGF receptor. Since cultured mesangial cells synthesize HB-EGF and this cytokine is a potent mitogen for smooth muscle cells (SMC), a cell type similar to mesangial cells, we attempted to determine (1) whether HB-EGF mRNA is present in the proliferative phase of experimental mesangial proliferative glomerulonephritis, and (2) some of the factors which regulate its synthesis by mesangial cells. In this study we demonstrate that cultured rat mesangial cells (RMC) express HB-EGF mRNA and that transcript levels are markedly increased by serum with maximal induction occurring within 2 h. Stimulation with individual cytokines (EGF, TGF-alpha, PDGF, TGF-beta and TNF-alpha), by contrast, had only a minor effect. The increase in HB-EGF mRNA levels following addition of serum was rapid, transient and independent of protein synthesis, features characteristic of immediate-early genes. In normal rat kidneys, there was no detectable glomerular expression of HB-EGF mRNA as determined by in situ hybridization, although occasional tubular cross sections were positive. Within 30 min after induction of the Thy-1.1 model, however, cells within the glomerulus and an increased number of tubules were positive. The number of positive glomerular and tubular cells increased progressively at days 1 and 4 post-induction, but declined by day 9 and had returned to background at day 15. Within the glomerulus, HB-EGF was expressed by cells of Bowman's capsule and cells within the glomerular tuft. Using (a) combined in situ hybridization and immunohistochemical staining of the same section and (b) staining of sequential sections by immunohistochemistry or in situ hybridization, it was found that intrinsic glomerular cells which did not stain with the macrophage marker ED-1 expressed HB-EGF mRNA. Although some were probably glomerular epithelial cells because of their peripheral location, it was uncertain whether mesangial cells were also positive. Future studies will be directed towards firmer identification of the glomerular cells expressing HB-EGF mRNA and to define the functional role of HB-EGF in the Thy-1.1 model.

Immunocytochemical colocalization of clusterin in apoptotic photoreceptor cells in retinal degeneration slow rds mutant mouse retinas.

In the rds mutant mouse the photoreceptor cells differentiate normally for the first few postnatal days, with the inner segments projecting an extended cilium. However, outer segments fail to form and only rudimentary disks and opsin-laden vesicles assemble at the tip of the cilium. These are shed into the interphotoreceptor space where they are phagocytosed by the retinal pigment epithelial cells. In this animal model, the photoreceptors undergo a slow degeneration via apoptosis leading to eventual loss of the entire photoreceptor population. Since increased expression of clusterin has been implicated in apoptosis, we studied the expression of clusterin in the rds mutant mouse retina and compared it to normal BALB/ c retinas. Small intestinal microvillus epithelium was used as a positive control tissue for apoptosis. Immunocytochemistry revealed the presence of clusterin in the ganglion cell, inner nuclear and outer plexiform layers and in the retinal pigment epithelium of both the rds and the BALB/c retinas. Interestingly, scattered clusterin-positive cells were observed in the outer nuclear layer (onl) of dystrophic retinas. Since the increased presence of clusterin protein in the onl of dystrophic retina may indicate dying photoreceptor cells due to apoptosis, we utilized a co-localization procedure for apoptotic nuclei and clusterin. For apoptosis we utilized an in situ 3' end labeling of fragmented DNA (TUNEL) and immunohistochemistry for clusterin using brown and red colored substrates respectively. Small intestine tissue sections were also included as positive controls for apoptosis. Our results show that clusterin is co-localized with apoptotic nuclei both in the onl of rds mutant retinas as well as in the small intestine epithelial cells undergoing cell turnover and exfoliation. These results are of interest since overexpression of clusterin is also observed in other neuro-degenerative diseases such as Alzheimer's and Pick's disease.

The use of anticlusterin monoclonal antibodies for the combined assessment of human sperm morphology and acrosome integrity.

Clusterin is an abundant protein in the human male reproductive tract which appears to be produced by the testis, epididymis and the seminal vesicles. Using monoclonal antibodies and an amplified immunoperoxidase technique, we have identified two apparently biochemically distinct forms of clusterin on human spermatozoa. Morphologically abnormal spermatozoa have an extensive surface coating of conventional 80 kDa native clusterin, but this form of clusterin is not detectable on normal spermatozoa. Normal spermatozoa, however, contain within the acrosomal cap a different form of clusterin, reactive with an anticlusterin alpha-chain antibody. Agglutinated spermatozoa, most of which are grossly abnormal, were intensely labelled with the antibody against conventional 80 kDa clusterin, suggesting that the 'clustering' properties of this protein may play a role in the aggregation of abnormal spermatozoa. Anticlusterin monoclonal antibodies may be useful for semen analysis. Staining spermatozoa with anticlusterin monoclonal antibodies is a technically simple method which provides a visually obvious means of assessing spermatozoa morphology and acrosome status simultaneously. The current data also suggest that different functions of clusterin in the reproductive tract may be attributed to different molecular forms of the protein.

Immunohistological localization of clusterin in the male genital tract in humans and marmosets.

Clusterin is a multifunctional protein, first described in the reproductive tracts of the rat and the ram. It is produced by several cell types and exists in at least two differentially glycosylated forms. The aim of this study was to extend knowledge of clusterin expression in the primate (human and marmoset) male reproductive tracts by means of clusterin-specific immunohistochemical techniques. In both normal and abnormal testicular tissue, clusterin was found in association with Sertoli cells, lumenal sperm, proacrosomal Golgi complexes, residual bodies, and degenerating germ cells. The major differences observed between the two groups were attributable primarily to morphological differences rather than to clusterin expression specifically. There was no correlation between testicular clusterin content and the cause and severity of spermatogenic disorders. Within normal epididymides, regional differences in clusterin staining similar to those reported in the rat were observed. The seminal vesicles contained large amounts of positive clusterin staining, whereas normal human prostate was completely negative. Low levels of clusterin expression were observed in the marmoset prostate. This study suggests that clusterin is an important and widespread product in the human and marmoset reproductive tracts and is likely to have a role in spermatogenesis.

Clusterin depletion enhances immune glomerular injury in the isolated perfused kidney.

Clusterin is a normal plasma protein, shown to be an inhibitor of reactive complement hemolysis and a component of the fluid phase SC5b-9 terminal complement complexes. It is a component of glomerular immune deposits in human and experimental glomerulonephritis. Using the complement-dependent isolated perfused rat kidney model of autologous phase passive Heymann nephritis, we have studied the effect of clusterin depletion of perfused plasma on the development of glomerular injury. Kidneys with planted glomerular sheep anti-rat Fx1A antibody were perfused with human plasma either depleted of clusterin to < or = 30%, or control plasma depleted of plasma fibronectin. Glomerular injury was then initiated by the addition of guinea pig anti-sheep immunoglobulins to the perfusate. Kidneys perfused with clusterin depleted plasma developed significantly greater proteinuria at all time points when compared to control kidneys. Glomerular antibody binding and C3 deposition were similar in the two groups, but terminal complement components were deposited in larger amounts in the clusterin depleted group. These data support a possible role for clusterin in vivo in the protection of complement-induced glomerular injury.

Abnormal distribution of retinal clusterin in retinitis pigmentosa.

Increased expression of clusterin mRNA is associated with neurodegenerative states, including retinas affected by retinitis pigmentosa (RP). We have investigated the distribution of immunoreactive clusterin in normal and RP-affected retinas. Reactivity at the inner limiting membrane, plexiform layers, and photoreceptors in normal retina accords well with clusterin's postulated role as a membrane protective agent. In RP-affected retina the organized distribution is lost and overall reactivity appears decreased. The changes in this case may reflect increased turnover or removal of clusterin, perhaps via interaction with components of the immune system.

Wegener's granulomatosis: clinical features and prognosis in 37 patients.

Thirty-seven patients (21 female, 16 male) with Wegener's granulomatosis (WG) were reviewed. Patients were followed for a mean six years after diagnosis; 14 were followed for more than seven years. The clinical features were similar to those in previous studies. In this series, only 13 patients (35%) had renal disease at presentation and the cumulative incidence of renal involvement was 51%. Thirty-one patients received treatment which included cyclophosphamide (CP). The case fatality rate of the six patients not treated with CP was 83% (five deaths). By contrast, all CP treated patients improved and 21 (68%) had complete remissions. Nine (29%) were in complete remission for a mean 4.9 years after discontinuing all treatment. Two were disease free for over ten years. The actuarial probability of survival for these patients was 97% at one year and 71% at ten years. Only three CP treated patients (10%) progressed to end-stage renal disease. The case fatality rate was 26% (eight patients) and sepsis was the cause of death in five. Fourteen patients (45%) treated with CP had at least one relapse of vasculitis and seven (23%) had multiple (two or more) relapses. These data indicate that CP is effective in inducing remissions and prolonging survival in patients with WG; however, relapses are frequent.