Isolation of plaice (Pleuronectes platessa) alpha1-microglobulin: conservation of structure and chromophore.
A cDNA coding for plaice (Pleuronectes platessa) alpha1-microglobulin (Leaver et al., 1994, Comp. Biochem. Physiol. 108B, 275-281) was expressed and purified from baculovirus-infected insect cells. Specific monoclonal antibodies were then prepared and used to isolate the protein from plaice liver and serum. Mature 28.5 kDa alpha1-microglobulin was found in both liver and serum. The protein consisted of an 184 amino acid peptide with a complex N-glycan in position Asn123, one intrachain disulfide bridge and a yellow-brown chromophore. Physicochemical characterization indicated a globular shape with a frictional ratio of 1.37, electrophoretic charge-heterogeneity and antiparallel beta-sheet structure. A smaller, incompletely glycosylated, yellow-brown alpha1-microglobulin as well as a 45 kDa precursor protein were also found in liver. The chromophore was found to be linked to alpha1-microglobulin intracellularly. Recombinant plaice alpha1-microglobulin isolated from insect cells had the same N-terminal sequence, globular shape and yellow-brown color as mature alpha1-microglobulin, but carried a smaller, fucosylated, non-sialylated N-glycan in the Asn123 position. The concentration of alpha1-microglobulin in plaice serum was 20 mg/l and it was found both as a 28.5 kDa component and as high molecular weight components. Thus, the size, shape, charge and color of plaice alpha1-microglobulin were similar to mammalian alpha1-microglobulin, indicating a high degree of structural conservation between fish and human alpha1-microglobulin. The monoclonal antibodies against plaice alpha1-microglobulin cross-reacted with human alpha1-microglobulin, emphasizing the structural similarity. (+info
The nuclear protein PH5P of the inter-alpha-inhibitor superfamily: a missing link between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and a novel actor of DNA repair?
Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick sensor involved in cellular processes where nicking and rejoining of DNA strands are required. The inter-alpha-inhibitor family is comprized of several plasma proteins that all harbor one or more so-called heavy chains designated H1-H4. The latter originate from precursor polypeptides H1P-H4P whose upper two thirds are highly homologous. We now describe a novel protein that includes (i) a so-called BRCT domain found in many proteins involved in DNA repair, (ii) an area that is homologous to the NAD-dependent catalytic domain of poly(ADP-ribose)polymerase, (iii) an area that is homologous to the upper two thirds of precursor polypeptides H1P-H4P and (iv) a proline-rich region with a potential nuclear localization signal. This protein now designated PH5P points to as yet unsuspected links between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and is likely to be involved in DNA repair. (+info
Temporal changes in mRNA expression for bikunin in the kidneys of rats during calcium oxalate nephrolithiasis.
Inter-alpha-inhibitor and other bikunin-containing proteins are synthesized in relatively large quantities by the liver. These proteins function as Kunitz-type serine protease inhibitors and appear capable of inhibiting calcium oxalate (CaOx) crystallization in vitro. Preliminary studies have shown that renal tubular epithelial cells synthesize bikunin in response to CaOx challenge. To examine this response in vivo, a sensitive reverse transcription-quantitative competitive template-PCR was developed to detect and quantify poly(A)+ -tailed bikunin mRNA expression in kidney tissue from normal rats and rats developing CaOx nephrolithiasis after challenge with ethylene glycol. Bikunin mRNA expression in rat liver tissue was assessed as a positive control. The expression of bikunin mRNA in liver did not differ significantly between normal control rats and experimental rats with induced hyperoxaluria and renal CaOx crystallization. In contrast, there were significant temporal increases in the levels of bikunin mRNA expression in rat kidneys during CaOx nephrolithiasis after challenge with ethylene glycol. Urinary excretion of bikunin-containing proteins seemed to increase concomitantly. These findings indicate an association between the induction of hyperoxaluria/CaOx nephrolithiasis and the expression of the bikunin gene in rat kidneys. (+info
Effect of long-term immunization against inhibin on sperm output in bulls.
To determine the effect of neutralization of inhibin on sperm output, 12 Holstein bulls were paired by birth date and weight on Day 1 of age. Each bull was actively immunized against bovine inhibin alpha1-26 gly-tyr (bINH) conjugated to human alpha globulin (HAG, n = 6 bulls) or HAG alone (controls, n = 6) at 60 days of age; booster immunizations were administered at 90, 104, 124, 270, and 395 days of age. Body weights and scrotal circumferences were measured at the time of primary immunization and at 10 days after each booster. In addition, jugular blood was obtained at 60, 70, 100, 114, 134, 280, and 405 days of age, during the 3-wk sperm collection period, and during a 6-h blood-sampling period after sperm collection to determine bINH antibody titer and concentrations of FSH, LH, testosterone, and estradiol. Beginning at 405 days of age, sperm output was measured 3 days/wk for 3 wk with two successive ejaculates collected each day for a total of 18 ejaculates per bull. During Days 60-405 of age, the increase in titer of bINH antibodies, scrotal circumference, and serum concentration of FSH was greater (p < 0.01) for the bINH-immunized compared with control bulls. There were significant (p < 0.01) pair x treatment interactions for sperm output and serum FSH and LH concentrations. Specifically, bINH-immunized bulls for four of the six pairs had nearly 50% greater serum FSH concentrations and sperm output. For the remaining two pairs, sperm output was lower and FSH was either lower or only marginally higher in the bINH-immunized bulls compared with controls. Also, the control bulls for the two remaining pairs produced more sperm than all but one bINH-immunized bull, and had markedly higher serum LH concentrations than all other bulls. To summarize, enhancement of sperm output after immunization against inhibin depends on the subsequent increment in FSH concentrations. We conclude that inhibin suppresses spermatogenesis. Thus, methods to immunoneutralize inhibin may have merit as a therapeutic route to enhance sperm production in reproductively maturing bulls. (+info
Glomerular and tubular proteinuria as markers of nephropathy in rheumatoid arthritis.
OBJECTIVE: We examined the prevalence of nephropathy in unselected patients with rheumatoid arthritis (RA) by measurement of marker proteins for glomerular and tubular damage in urine. METHODS: A highly sensitive immunoluminometric assay was used to measure albumin, immunoglobulin G and alpha1-microglobulin in 24 h urines of 44 RA patients and a control group of 46 patients with generalized osteoarthritis (OA). RESULTS: Fifty-five per cent of RA patients were found to have proteinuria as a symptom of renal disease. Drug therapy or vasculitis were identified as possible reasons for proteinuria in only 25% of these patients; in most patients (75%), no reason for proteinuria was found. Tubular and mixed proteinuria were more frequent than glomerular proteinuria. Only 15% of the control group exhibited mild proteinuria, which was attributable to nephrotoxic factors. The renal function of RA patients and the control group did not differ significantly. CONCLUSIONS: Proteinuria is a frequent symptom of nephropathy in RA. Screening for renal disease in RA should not only include creatinine measurement and dipstick examination of urine, but also more sensitive methods to detect tubular and glomerular proteinuria as a marker of tubular and early stages of glomerular damage. (+info
Isolation and characterization of proteoglycans from human follicular fluid.
Two proteoglycans differing in size and composition were isolated from human follicular fluid. The larger one of high density had a molecular mass of 3.0x10(6) Da, as determined by laser light-scattering, and was substituted with 15-20 chondroitin sulphate (CS) chains (Mr 60000-65000). Half of the CS disaccharides were 6-sulphated, whereas the remaining ones were non-sulphated. Digestion of the CS proteoglycan with chondroitinase ABC lyase, followed by SDS/PAGE, yielded a protein core of 600 to 700 kDa including substituted oligosaccharides, and a band of 70 kDa that was identified as the heavy-chain component of the inter-alpha-trypsin inhibitor (ITI). Western blotting of the CS proteoglycan showed that this had reactivity with antibodies raised against human versican. Electron microscopy (EM) of the CS proteoglycan also revealed a versican-like structure, with one globular domain at each end of a long extended segment substituted with CS side chains, as well as a structure interpreted as being the heavy chain of ITI attached to CS chains. Laser light-scattering revealed that the smaller proteoglycan had a molecular mass of 1. 1x10(6) Da, and EM demonstrated that it had a globular-protein core structure. The core protein, which showed immunological reactivity with perlecan antibodies, was substituted with approximately seven heparan sulphate (HS) and CS chains of similar size (50-55 kDa), the CS disaccharides being mainly 6-sulphated (68%), with a small proportion being 4-sulphated. The protein core was shown to be heterogeneous, with bands occurring at 215, 330 and 400 kDa after enzymic degradation of the glycosaminoglycan chains followed by SDS/PAGE analysis. The demonstration of intact molecules and fragments obtained after stepwise degradations, as shown by gel chromatography, supported a 'composite' structure of this proteoglycan. (+info
Renal involvement in Gambian children with cerebral or mild malaria.
Kidney function was studied in 80 Gambian children with cerebral malaria, 73 children with mild malaria, and in 19 children with other febrile illnesses. Serum creatinine was measured, and the excretion in urine of immunoglobulin G, transferrin, albumin and alpha 1 microglobulin was determined. Twenty-five percent of children with cerebral malaria, and 4% of children with mild malaria had an elevated serum creatinine above 62 mumol/l. Increased urinary protein excretion was frequent: 53% of children with cerebral malaria had a glomerulo-tubular pattern of protein excretion, and 46% a tubular pattern. Median albuminuria was 68 mg/l in children with cerebral malaria, 18 mg/l in children with mild malaria, and 9 mg/l in febrile children with other diseases (P < 0.0001). There was no significant association between the proteinuria and height of fever or the degree of parasitaemia, and there was no significant association between death and signs of renal impairment. Renal involvement is common in children with malaria in The Gambia, with prerenal, glomerular, and tubulo-interstitial factors contributing. It is more pronounced in children with cerebral malaria than in those with mild malaria. However, renal dysfunction is relatively mild and does not indicate a worse prognosis. (+info
Inter-alpha-inhibitor binding to hyaluronan in the cumulus extracellular matrix is required for optimal ovulation and development of mouse oocytes.
This report characterizes the effects of excess hyaluronan (HA) upon the expansion of the cumulus oocyte complex (COC) within intact follicles and upon ovulation and oocyte viability in mice. Covalent linkage between heavy chains of the inter-alpha-inhibitor (IalphaI) family of serum glycoproteins and HA is necessary for optimal cumulus extracellular matrix (cECM) stabilization and cumulus expansion. Intravenous administration of HA oligosaccharides inhibited the binding of IalphaI to endogenous HA, disrupting the process of expansion and resulting in a reduction in the size of the cumulus mass. Western blot and immunocytochemical analyses of COCs from HA-treated animals demonstrated a reduction of IalphaI heavy chains within the cECM. Additionally, HA-treated immature animals ovulated 56.3% fewer COCs compared to control animals. The developmental potential of COCs in HA-treated animals was also tested. Extended periods of oviductal storage of COCs ovulated by HA-injected adult mice resulted in a reduction of normal embryos and a significant increase in the proportion of fragmented oocytes/embryos. These observations support the view that covalent binding of IalphaI heavy chains to HA is required for optimal cumulus expansion, extrusion of the COCs from the follicle at ovulation, and maintenance of oocyte viability within the oviduct. (+info