Identification of possible regions of chaperone activity in lens alpha-crystallin.
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Animals↗
Biomedical subjects
J B Smith
Publications and source records attributed to J B Smith.
Modifications of the water-insoluble human lens alpha-crystallins.
Since the water-insoluble crystallins of the lens may be the precursors of cataract, identifying the modifications that differentiate the water-insoluble from the water-soluble crystallins may provide the basis for understanding the chemistry leading to cataract. This investigation of the alpha-crystallins of the water-insoluble urea-soluble portion of 45-year-old normal clear lenses, isolated using gel filtration, ion exchange and reversed phase chromatography, has employed state-of-the-art mass spectrometric techniques to identify and locate the modifications of the water-insoluble alpha-crystallins. Modifications present in the isolated alpha-crystallins were identified by the molecular weights of the modified proteins, by the molecular weights of peptides produced by enzymatic digestion of the proteins, and by the fragmentation patterns produced by collisional activation of the peptides. Modifications that are either unique to the water-insoluble alpha-crystallins or are more prevalent in the water-soluble portion than in the water-soluble part include complete oxidation of the two Cys residues of alpha A-crystallin to form an intra-molecular disulfide bond, partial truncation at both the C-termini and N-termini of alpha A- and alpha B-crystallins, partial oxidation of Met residues to methionine sulfoxide, partial deamidation of several Asn and Gln residues, and evidence of peptide bond cleavage at some of the deamidated residues. Although many reactions have been proposed to contribute to the insolubility of crystallins, this compilation of in vivo post-translational modifications of water-insoluble alpha-crystallins delineates products that are actually present at levels of 5% or more. From these results, it is hypothesized that alpha-crystallin becomes water-insoluble following deamidation of various Asn and Gln residues which cause conformational changes leading to formation of an intra-molecular disulfide bond between the Cys residues of alpha A-crystallin.
Amino Acid Sequence↗
Cutaneous manifestations and consequences of smoking.
Cigarette smoking is strongly linked to serious internal diseases such as cancer, cardiovascular disease, and lung disease. However, the external manifestations and consequences of smoking are relatively unknown. Although generally less ominous, the cutaneous manifestations of smoking may be associated with significant morbidity. This article reviews the known adverse effects on the skin of smoking.
Humans↗
Pyoderma gangrenosum in a patient with cryoglobulinemia and hepatitis C successfully treated with interferon alfa.
We describe a patient with long-standing pyoderma gangrenosum unresponsive to therapy. The patient had concomitant cryoglobulinemia and hepatitis C. When the hepatitis C was treated with interferon alfa-2a his pyoderma gangrenosum resolved. Whether this was from the interferon alfa or spontaneous resolution is not known.
Adult↗
Reflex sympathetic dystrophy with mutilating ulcerations suspicious of a factitial origin.
Reflex sympathetic dystrophy (RSD) is a poorly understood posttraumatic pain syndrome associated with dysfunction of the sympathetic nervous system. Pain is often out of proportion to the extent of injury. Progression of the disease may lead to dystrophic and atrophic changes resulting in total disability of an affected limb. Skin findings are highly variable and nonspecific and may rarely include bullae and ulcerations. We describe a mutilating case of RSD with unusual and severely disfiguring ulcerations that necessitated amputation of the right arm. Shortly after the amputation, ulcerations began appearing on the left arm. We suspect a factitial component but have been unable to prove or disprove it. We propose that some ulcerations in patients with RSD may be factitial in origin.
Female↗
Circle hairs are not rolled hairs.
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Aged↗
Rapid assessment of early glycation products by mass spectrometry.
In order to detect the early glycation products, we have reacted a model peptide (t-boc-lys-ala-ala) with L-threose (a degradation product of ascorbic acid) and analyzed the reaction products by a combination of HPLC and mass spectrometry. Amino group modification, as observed by a fluorescamine assay, indicated complete modification after 3 days of incubation with a 10-fold excess of threose. As much as 60% of the adducts were acid labile and only 4% of the adducts could be observed by amino acid analysis. However, Fast atom bombardment mass spectrometry (FABMS) of the samples incubated for 6 hr showed relative molecular masses consistent with the formation of adducts corresponding to the addition of one and two molecules of L-threose to the peptide. Likewise, samples incubated for 12 hr showed peptide adducts with two and three L-threoses. The number of threose molecules added to the peptide was also confirmed from the FABMS analysis by using [1-13C]-threose as the glycating agent.
Carbon Isotopes↗
Calcium bioavailability from bovine milk and dairy products in premenopausal women using intrinsic and extrinsic labeling techniques.
Stable isotopes were used to compare calcium fractional absorption from intrinsically and extrinsically labeled bovine milk as well as intrinsically labeled dairy product and cheese analogue. Healthy Caucasian women were fed a controlled diet for 4 d during the follicular phase of their menstrual cycle. With breakfast on the third day, participants ingested milk containing 44Ca (intrinsic) and 42CaCl2 (extrinsic) or dairy products containing 44Ca. Total feces were collected for 2 d prior to and 10 d after isotope ingestion. Polyethylene glycol was administered to monitor completeness of fecal collections. Total calcium was determined by atomic absorption spectrophotometry, and isotopic abundance was determined by high resolution fast atom bombardment mass spectrometry. Fractional absorption was determined as the difference between the administered isotopic dose and the quantity of 44Ca or 42Ca excreted in feces. The fractional absorption of calcium from milk was not affected by the method of labeling, lactose content, fermentation or the chemical form of calcium in dairy products or cheese analogue.
Absorption↗
Prenatal diagnosis using fetal cells isolated from maternal peripheral blood: a review.
Many questions remain about the feasibility of using fetal cells from maternal blood for prenatal diagnosis. Although recently there has been more focus on clinically relevant methods, many studies have been performed using blood drawn after invasive procedures, and over a wide range of gestational ages. For methods to be applicable to clinical use, more work is needed on isolating cells early in pregnancy, when termination is still an option for parents who are found to have an affected pregnancy. It is generally agreed that fetal nucleated erythrocytes are the most efficacious cell type for prenatal diagnosis, but it has not yet been shown definitively whether there is an ideal gestational age for sampling, whether ABO incompatibility might limit availability of fetal cells, or whether the number of cells present might be different in normal versus abnormal pregnancies. PCR has been shown to be a powerful tool in allowing amplification and identification of very small amounts of fetal DNA. However, this is limited to cases in which a specific and unique gene from the father is sought. This means that there is the potential to diagnose many paternally inherited autosomal dominant diseases and some autosomal recessive diseases, in which the parents have different and identifiable mutations. However, when parents are both carriers of the same autosomal recessive mutations, or when the disease is X linked, PCR will not aid in prenatal diagnosis. Cytogenetic analysis of fetal cells by FISH after cell sorting is another potentially useful method of prenatal diagnosis, but requires relatively pure samples of fetal cells or an independent marker that allows easy microscopic identification. The latter might be accomplished by identifying fetal cells through their expression of embryonic hemoglobins or because they contain HLA-G mRNA. In addition, current techniques of cell sorting must be improved so that a higher percentage of fetal cells can be isolated. Currently, the best cell sorting techniques usually produce a maximum purity of 10% fetal cells. Commonly, in normal pregnancies, fewer than 0.1% of the cells isolated after sorting are fetal in origin. Improving the concentration and quantity of fetal cells will improve the accuracy of FISH. Methods such as immunophenotyping that allow the selective identification of fetal cells by microscopy, and can be used in conjunction with FISH, may be extremely valuable because they may allow the genetic analysis of only the few fetal cells within a background preponderance of maternal cells. Although the retrieval of fetal cells from maternal blood is an attractive concept, it must be clearly stated that presently it is only in the investigational phase because of the low sensitivity and specificity. There is no current application for these methods in clinical practice. It remains to be determined whether testing maternal blood for fetal cells or DNA will be used as a screening tool, similar to the maternal serum screening currently in use, or whether the accuracy can be improved to a level such that the techniques can be used diagnostically. Although there are many questions that remain unanswered at this time, the outlook for noninvasive prenatal genetic testing in the future is optimistic.
Erythrocytes↗
Maternal morbidity among women admitted for delivery at a public hospital in Kathmandu.
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Asia↗
Calcium homeostasis in smooth muscle cells.
Calcium triggers muscle contraction and is a second messenger of hormones and growth factors that regulate metabolism, gene expression, and secretion in smooth muscle cells (SMC). SMC contain dozens of proteins that bind Ca2+ either to buffer changes in ionized calcium or to elicit a cellular response such contraction or secretion. Although there practically an infinite supply of Ca2+ in extracellular medium, SMC respond to a variety of stimuli by mobilizing Ca2+ accumulated in the sarcoplasmic reticulum (SR), where most cellular Ca2+ resides. The SR of smooth muscle resembles the endoplasmic reticulum of nonmuscle cells and accumulates Ca2+ via the sarcoendoplasmic reticulum Ca2+ adenosine triphosphatase (ATPase). Stimuli, such as angiotensin II, produce inositol 1,4,5,-trisphosphate (IP3), which regulates a Ca2+ channel of the SR. IP3 binding opens the channel and produces a "spike" in the cytoplasmic concentration of free Ca2+ ([Ca2+]i). The spike is largely due to the release of stored Ca2+ because hormonal stimulation produces similar spikes in the presence and absence of extracellular Ca2+. Ca2+ ejection from the cell, rather than reaccumulation by the SR, is responsible for rapidly decreasing [Ca2+]i from the peak level produced by the stimulus. Release of SR Ca2+ and activation of plasma membrane Ca2+ efflux mechanisms markedly decrease total cell Ca2+. Two independent Ca2+ transporters in the plasma membrane, the Na(+)-Ca2+ exchanger and the Ca2+ ATPase, actively eject Ca2+ from SMC. The Na(+)-Ca2+ exchanger is largely responsible for the acute phase of Ca2+ ejection, whereas the plasma membrane Ca2+ ATPase contributes to the sustained increase in Ca2+ efflux from stimulated SMC. Following Ca2+ release from the SR and ejection from the cell, Ca2+ enters via channels, which sustain a modest increase in [Ca2+]i and a gradual refilling of the SR. Mitochondria have an important role in intracellular Ca2+ signaling. Mitochondrial metabolism is highly responsive to transient increases in [Ca2+]i, although mitochondria are not a Ca2+ repository. Ca2+ uptake by mitochondria is driven by the highly favorable electrochemical potential difference across the inner membrane. Mitochondria actively expel Ca2+ via a H(+)-Ca2+ or Na(+)-Ca2+ exchanger. Ca2+ uptake and ejection by mitochondria contributes to temporal and spatial oscillations in [Ca2+]i. accelerated Ca2+ cycling between the Sr, cytoplasm, mitochondria, and the environment is a hallmark of cell stimulation.
Animals↗
The effect of reduced extrinsic sucrose intake on plasma triglyceride levels.
OBJECTIVE: To examine the effect on plasma triglycerides of removing most of the extrinsic sugar from the diet of free living hypertriglyceridaemic individuals while controlling weight loss. DESIGN: Free living subjects were randomly assigned to intervention and control groups. The intervention group undertook to consume a "sugar free' diet for six months after a three month baseline period. The control group maintained their usual diets for the nine month period. The results from the two groups were compared using repeated measures ANOVA. SUBJECTS: Thirty two hypertriglyceridaemic individuals, selected from members of the public in Dunedin, New Zealand. RESULTS: Subjects in the intervention group were able to achieve a mean reduction in sucrose intake of > 70%, largely extrinsic sucrose, resulting in a group mean reduction in plasma triglyceride levels of > 20% and a mean weight loss over the six month period of about 2%. Repeated measures ANOVA on the mean triglyceride figures after the individual figures had been adjusted for this small weight loss, showed that there was a significant fall in triglyceride levels over the study period compared with the baseline. CONCLUSIONS: Hypertriglyceridaemia can be reduced in some individuals by the reduction of extrinsic sucrose in the diet. When allowance is made for weight loss the reduction is still significant.
Adult↗
Treatment of rheumatoid arthritis by immunization with mononuclear white blood cells: results of a preliminary trial.
OBJECTIVE: To determine if immunization with alloantigenic blood mononuclear cells (MNC) in active rheumatoid arthritis (RA) can result in objective and subjective improvement in RA. METHODS: Eleven patients meeting American College of Rheumatology criteria for RA were treated in an open label trial by immunization with allogeneic MNC obtained from donors screened for infectious agents according to American Association of Blood Banks guidelines. MNC (30-250 x 10(6) cells) were given at 6 week intervals. Half the MNC (vol 2 ml) were injected intravenously and half in 4 divided doses subcutaneously (sq, 0.5 ml each). Disease activity assessments were done before entry and at immunotherapy visits thereafter. These included physician global assessment of disease activity, patient global assessment of pain, arthritis impact measurement scales (AIMS), swollen joint count, erythrocyte sedimentation rate (ESR), and/or C-reactive protein (CRP). RESULTS: Statistically significant improvement was noted in all clinical variables measured when pretreatment data were compared to those obtained at the time of injection number 3. MNC dose related improvement was found when the total number of cells given in the first 2 injections were compared to percentage improvement in patient assessment of pain (r = 0.71, p < 0.02), AIMS (r = 0.60, p = 0.05), and improvement in the means of all variables measured (r = 0.70, p < 0.03). When all variables were averaged, 6 of 11 patients experienced > 20% and 3 of 11 experienced > or = 40% improvement. The only side effects noted were transient local pain and swelling at the sq injection sites. CONCLUSION: MNC immunization may represent a suitable and safe alternative to drug treatment for selected patients with RA. Statistically significant improvement was found in all variables and several of these were cell dose related. A placebo controlled randomized trial immunizing with a standardized number of cells will address efficacy of this biological treatment approach.
Adult↗
Glucocorticoid-attenuated response genes encode intercellular mediators, including a new C-X-C chemokine.
A major part of the anti-inflammatory effect of glucocorticoids is attributable to their attenuation of the induction of genes whose products mediate intercellular interactions, e.g. cytokines and the inducible forms of prostaglandin synthase and nitric oxide synthase. We hypothesized that (i) there exists a class of immediate-early/primary response genes whose induction by inflammatory agents, mitogens, and other stimuli is attenuated by glucocorticoids, and (ii) the products of these glucocorticoid-attenuated response genes (GARGs) function predominantly in paracrine cell processes. We constructed a lambda cDNA library from transforming growth factor beta 1-pretreated murine Swiss 3T3 cells stimulated with lipopolysaccharide (LPS) or serum in the presence of cycloheximide, screened 15,000 plaques by differential hybridization, and cloned 12 LPS-induced, dexamethasone-attenuated cDNAs. Seven were previously known. Six of these encode intercellular mediators (thrombospondin-1, MCSF, JE/MCP-1, MARC/fic/MCP-3, crg2/IP-10, and cyr61); one encodes a protein of unknown function (IRG2). Thus, a large majority of these GARG cDNAs encode intercellular mediators, as hypothesized. Of the five GARG cDNAs not previously known, one encodes a novel member of the CXC chemokine family, designated LIX (LPS-induced CXC chemokine). The predicted LIX protein has a 40-amino acid signal sequence and a 92-amino acid mature peptide with a distinctive COOH-terminal region. Surprisingly, segments of the 3'-untranslated regions of LIX and two other CXC chemokines have substantially greater nucleotide sequence homology than do their coding regions. These segments may perform an unknown regulatory function. The LIX message is strongly induced by LPS in fibroblasts, but not in macrophages, suggesting that LIX may participate in the recruitment of inflammatory cells by injured or infected tissue.
3T3 Cells↗
The complete sequence of human lens gamma s-crystallin.
The complete sequence of human gamma s-crystallin has been determined and confirmed using a combination of MS methods, peptide sequencing and cDNA sequencing. Regions 21-35 and 102-107, which were previously assumed to be the same as the bovine sequence, differ from the bovine sequence at residues 22, 28, 31 and 104. An additional six residues were also found to be different from the original sequence determined for Pakastani lenses. Whether these differences represent errors in the original sequence or two different sequences among human lens crystallins is not yet known.
Amino Acid Sequence↗
Molecular cloning and expression of catrocollastatin, a snake-venom protein from Crotalus atrox (western diamondback rattlesnake) which inhibits platelet adhesion to collagen.
A 50 kDa protein that inhibits platelet adhesion to collagen has been isolated from snake venom of Crotalus atrox (western diamondback rattlesnake) and has been named 'catrocollastatin'. The cDNA cloning of catrocollastatin has been accomplished. A full-length cDNA of 2310 bp with an open reading frame between nucleotides 51 and 1880 was obtained. The deduced amino acid sequence consists of 609 amino acids. The cDNA-predicted amino acid sequence is highly similar to that of haemorrhagic metalloproteinase jararhagin from Bothrops jararaca venom, HR1B from Trimeresurus flavoviridis, Ht-e from C. atrox and trigramin from T. gramineus. Like jararhagin and HR1B, catrocollastatin is a multidomain molecule composed of an N-terminal domain, a metalloproteinase domain, a disintegrin-like domain and a cysteine-rich C-terminal domain. In the disintegrin-like domain, the frequently seen RGD (Arg-Gly-Asp) sequence is replaced by SECD (Ser-Glu-Cys-Asp). This cDNA was expressed in Spodoptera frugiperda (fall armyworm) (Sf9) insect cells using a baculovirus expression system. Like native catrocollastatin, the expressed protein is capable of selectively blocking collagen-induced platelet aggregation. This is the first full-length clone of a high-molecular-mass haemorrhagin to be expressed.
Amino Acid Sequence↗
Glutathione adducts, not carbamylated lysines, are the major modification of lens alpha-crystallins from renal failure patients.
alpha-Crystallins from the water-soluble and the water-insoluble, guanidine-soluble portions of lenses from four renal failure patients and two normal donors of similar age were isolated and enzymatically digested into peptides. Molecular weights of the peptides, determined by fast atom bombardment mass spectrometry, indicated modifications specifically associated with renal failure. The only modifications observed in the alpha-crystallins from renal failure patients, but not in the normal old lenses, were glutathione adducts to Cys 131 and Cys 142. These adducts were present in the lenses of all four renal failure patients, but not in the two normal old lenses. The four lenses from the renal failure patients were searched for evidence of carbamylation at lysyl or cysteinyl residues: carbamylation was not detected. Because the same mass spectrometric methods had previously demonstrated sufficient sensitivity and specificity to detect as little as 5% modification in the examination of in vitro carbamylated bovine lenses, these results indicated that carbamylation is not a major modification of the lens alpha-crystallins of renal failure patients.
Aged↗