Human Globulin Research Articles

Labeling of Proteins with [35S]Methionine and/or [35S]Cysteine in the Absence of Cells

Incubation of [35S]methionine and [35S]cysteine with bovine albumin, globulin, catalase, hemoglobin, or human globulin resulted in incorporation of the35S label into each of these proteins. Trichloroacetic acid (TCA) precipitation revealed that the percentage of label incorporated ranged from 1 to 15%. The35S labeling was resistant to dissociation by reducing SDS–PAGE, prolonged dialysis against 4 M urea, heating, TCA precipitation, and dilution by gel filtration. The labeling effect was more efficient with [35S]cysteine than [35S]methionine. Incubation of35S label with proteins differing in methionine and cysteine content revealed no requirement for sulfur-containing amino acids in the target protein. Protein carboxymethylation reduced but did not prevent35S label incorporation. Amino acid analysis of labeled proteins revealed that the radioactive label was not consistently associated with an individual amino acid. Differences in the ability of various proteins to spontaneously label with these amino acids suggest caution in the interpretation of metabolic labeling experiments and the necessity for inclusion of additional controls. Alternatively, our experience indicates a potentially useful method for labeling proteins in the absence of cells.

The mucous neck cell in the human gastric corpus: a distinctive, functional cell lineage.

There is considerable debate about whether the mucous neck cell (MNC) in the mucosa of the gastric corpus is merely a transit cell population, intermediate between gastric stem cells and the differentiated zymogenic (chief or peptic) cell lineages, or has distinct functions of its own. To cast light on these possibilities, the secretory phenotype of the MNC has been examined. Archival gastric body samples from non-ulcer dyspepsia biopsies and gastrectomies performed for peptic ulcer disease were stained with antibodies to the trefoil peptides TFF1/pS2 and TFF2/SP, pancreatic secretory trypsin inhibitor (PSTI), epidermal growth factor (EGF) and its receptor (EGFR), and to the MUC1 gene product--HMFG2. Human MNCs express PSTI, TFF1/pS2, TFF2/SP, and EGF proteins, while rat MNCs express TFF2/SP; the mucin contained in the MNCs is diastase/periodic acid Schiff (D/PAS)-positive and stains with human milk fat globulin (HMFG2). The canaliculi but not the cytoplasm of adjacent parietal cells were also decorated focally by D/PAS, by HMFG2, and by antibodies to TFF2/SP and TFF1/pS2. These findings favour the hypothesis that MNCs have a defined phenotype and are thus a separate and distinct cell lineage, secreting a number of luminally-active peptides which protect the gastric mucosa, and in particular the adjacent parietal cells, from the effects of secreted gastric acid. Moreover, a considerable degree of similarity in secretory profile is noted between MNCs and the so-called 'reparative lineages' in the gut--the ulcer-associated cell lineage (UACL) and hyperplastic polyp epithelium. If, on the other hand, the MNCs are indeed a transit population differentiating into zymogenic or peptic cells, then it is clear that having differentiated into one secretory phenotype producing a range of peptides, the MNC then proceeds to differentiate into a cell with a totally different secretory phenotype, a phenomenon unique in gastrointestinal cell lineage relationships.

Globulins in protein supplements promote the development of preimplantation embryos.

Our purpose was to investigate the effect of alpha- and beta-globulins contained in protein supplements on the development of preimplantation embryos. Mouse one-cell embryos were cultured in medium supplemented with 4 mg/ml human serum albumin (HSA), 4 mg/ml HSA plus human globulins (0.2, 0.4, 0.8, and 1.6 mg/ml) that consisted predominantly of alpha- and beta-globulins, or 10% Plasmanate Cutter (PC). Blastocysts developed in media supplemented with these various protein sources were stained with Hoechst 33342 to determine the number of cells. Supplementation with 0.4 to 1.6 mg/ml globulins or PC significantly increased the rate of blastocyst development compared with that observed with the addition of HSA. Supplementation with globulins significantly increased the hatching rate in a dose-dependent manner. The number of cells in the blastocysts was significantly increased when the embryos were cultured with 0.8 mg/ml of the globulins or PC. The present observations suggest that alpha- and beta-globulins in protein supplements promote embryo development and hatching.

Rem syndrome reproduced in UVA cabin

CD44 is a polymorphic integral membrane glycoprotein that serves as the principal cell surface receptor for hyaluronate, the major component of the extracellular matrix. CD44 is abundantly found in the skin and functions as a cell adhesion molecule. In a recent study we have observed a massive dermal accumulation of hyaluronate as a result of the in vivo selective suppression of CD44 in keratinocytes in mice expressing a keratin 5 promoter-driven CD44 anti-sense transgene. As the histologic features of the dorsal skin of these transgenic mice display some similarities to those of the skin lesions of lichen sclerosus et atrophicus, we explored the nature of the material accumulated in the dermis of genital and extragenital lesions of 14 patients with lichen sclerosus et atrophicus by Alcian Blue and human CD44 receptor globulin stainings, as well as the epidermal expression of CD44 protein and mRNA by immunohistochemistry and in situ hybridization. In this study we provide evidence that hyaluronate is accumulated in the superficial dermis of lichen sclerosus et atrophicus lesions, in particular by the use of human CD44 receptor globulin staining, which binds specifically to hyaluronate. In addition we show that the protein and mRNA expression of CD44 in the epidermis of the involved lichen sclerosus et atrophicus skin from genital and extragenital areas is significantly decreased, and in some cases completely lost. In contrast, keratinocyte CD44 expression was un-altered in the skin lesions of lupus erythematosus, scleroderma and reticular erythematous mucinosis, despite the presence of a mucinous material in the dermis. These results suggest that a decrease in CD44 in the keratinocytes may be correlated with an abnormal dermal accumulation of hyaluronate in the lesions of lichen sclerosus et atrophicus, and may play a pathogenetic role in this disease.

Agammaglobulinemia, by Col. Ogden C. Bruton, MC, USA,Pediatrics, 1952;9:722–728

A hitherto unrecognized entity manifested by complete absence of gamma globulin with otherwise normal serum proteins and recurrent pneumococcal sepsis is described in an 8-year-old male. The patient appeared to be normal in other respects and, after extensive study, no structural or functional change could be demonstrated in any body system. He was unable to produce antibody to the pneumococcus with the four antigenic substances used; a positive Schick test persisted despite numerous attempts to reverse it with diphtheria toxoid. No antibody could be demonstrated following administration of typhoid vaccine in the usual manner, and his serum was negative for complement-fixing antibodies of epidemic parotitis after he experienced a typical clinical picture of that disease.Gamma globulin could be demonstrated in his serum after concentrated immune human serum globulin was administered subcutaneously, and its gradual disappearance could be followed by electrophoretic analysis over a period of approximately six weeks. Concurrently, and following administration of human gamma globulin (3.2 gm. gamma globulin) at monthly intervals, he had been free of pneumococcal sepsis for more than a year, whereas he had experienced clinical sepsis at least 19 times in the previous four years. Eight different types of pneumococci had been recovered from blood cultures during 10 different episodes of sepsis.In the Discussion, [the author] concluded that there was a cause-and-effect relationship between the absence of gamma globulin and the repeated infections, based on the child's dramatic improvement after beginning gamma globulin therapy. [The author] proposed two possible causes–congenital or acquired. While [he] felt that the patient's good health for the first 4.5 years of life argued against a congenital cause, the persistence of the defect supported it.

Respiratory syncytial virus immunoglobulin and monoclonal antibodies in the prevention and treatment of respiratory syncytial virus infection

Respiratory syncytial virus (RSV) infection is particularly severe in infants with bronchopulmonary dysplasia (BPD) and in premature infants without BPD. Attempts to develop a vaccine against RSV have been unsuccessful. Passive immunoprophylaxis of premature infants with or without BPD using a hyperimmune human globulin against RSV (RSVIg) decreases the severity of RSV infection such that the rate of hospitalization following infection is reduced by 40%. The severity of illness among hospitalized infants is also reduced. To avoid the difficulties associated with intravenous infusion of immunoglobulins, monoclonal IgG antibodies against the fusion protein of RSV have been developed. In one trial, the antibodies were ineffective, although subsequent trials using higher doses of the antibody show more promising results. Studies of IgA monoclonal antibodies directed against RSV, which are administered in the form of nose drops, are also in progress. None of these preparations appear to be effective in the treatment of established RSV infection. Each of the preparations appeared to be safe, with one exception. Infants with cyanotic heart disease who received RSVIg had an increased risk of surgical complications, perhaps related to increases in serum viscosity as a result of receiving the hyperimmunoglobulin monthly in doses of 750 mg/kg. While definitive cost/benefit analyses are pending, RSVIg may be useful in infants and children with BPD who have current or recent oxygen requirements, as well as in certain premature infants without BPD. Recommendations on the use of RSVIg are provided.

Development and characterization of new immortalized human breast cell lines

New human breast cell lines were developed from metastatic breast cancer tissues and normal breast tissues. Primary cultures were initiated from cellular outgrowths of explanted tissues or from mechanically isolated cells in two serum-free media. Cell cultures derived from both cancer and normal tissues were immortalized with pRSV-T plasmid to generate permanent breast cell lines that exhibited an epithelial morphology. Cell lines generated in this study were characterized with respect to morphology, growth rate, karyotype, presence of specific genes, and the expression of epithelial and breast markers. The cell lines expressed the epithelial cell markers, cytokeratins 8 and 18, and retained the capacity to produce human milk fat globulin. They also express the BRCA-1, erbB2, and EGF receptor genes and possess the H-ras, K-ras, and p53 genes. Preliminary data showed that one of the new cancer cell lines was highly sensitive to the cytotoxic action of taxol. It is envisioned that the new breast cell lines will be useful as targets for identification of therapeutic agents against breast cancer and as models for carcinogenesis studies.

Separation of components of human globulins by capillary zone electrophoresis using a linear polyacrylamide-coated capillary

Eleven commercially available protein preparations from human serum were subjected to capillary zone electrophoresis (CZE) using a linear polyacrylamide-coated capillary at pH 7.4. Transferrin, complement C3 and C-reactive protein were each separated into one major peak and several minor peaks. α 1-Antitrypsin was separated into two major peaks and three minor peaks. α 2-HS-glycoprotein showed four major peaks with a leading shoulder. Haptoglobin, α 2-microglobulin, α 1-acid-glycoprotein and prealbumin were detected as relatively wide peaks. Ceruloplasmin showed one major peak with notches, and two minor and several notched peaks. Only low density lipoprotein showed no peaks. A mixture of five of the protein preparations was separated into individual components, as well as individual isoforms. When the same mixture was analyzed by CZE using an uncoated capillary, a much poorer resolution was obtained. Application of this CZE system to albumin-depleted serum demonstrated that it is very useful for analyzing globulin components in serum.

Respiratory Syncytial Virus Immune Globulin

Respiratory syncytial virus (RSV) immune globulin is a purified human hyperimmune globulin that provides passive immunity against complicated RSV disease in select groups of infants and young children. According to microneutralisation assay results, its RSV-neutralising antibody concentration was significantly greater than that of nonspecific immune globulin, thereby suggesting the potential for more reliable protection against RSV infection. In 2 randomised double-blind trials, prophylaxis with RSV immune globulin significantly reduced (vs no immune globulin) the incidence and severity of RSV disease in infants and young children with bronchopulmonary dysplasia, prematurity or bronchopulmonary dysplasia due to prematurity, or congenital heart disease (in 1 study). Treatment with RSV immune globulin did not significantly reduce the duration of hospitalisation and intensive care in children hospitalised with RSV infection in 2 randomised double-blind trials; however, a trend towards a significant treatment benefit was apparent in children with severe disease in 1 of these studies.

Apocrine cystadenoma, apocrine hidrocystoma, and eccrine hidrocystoma: three distinct tumors defined by expression of keratins and human milk fat globulin 1.

Eccrine hidrocystomas and apocrine cystadenomas are morphologically related cystic sweat gland tumors. To elucidate their cellular differentiation we examined by immunohistochemistry the expression of keratins and of human milk fat globulin 1 in 12 of each of these tumors, diagnosed using established conventional histological criteria. All tumors diagnosed as apocrine cystadenomas by these criteria were characterized by a keratin pattern of secretory type. In addition, they expressed human milk fat globulin 1. Tumors diagnosed as eccrine hidrocystomas expressed a keratin pattern of excretory type. A part of the tumors with an excretory keratin pattern expressed human milk fat globulin, while others did not. Some presumed eccrine hidrocystomas expressed the very same antigens as apocrine cystadenomas. Thus, our study reveals three distinct types of tumors, in contrast to the conventional distinction of only eccrine hidrocystomas and apocrine cystadenomas. Apocrine cystadenomas differentiate towards the secretory coil of apocrine sweat glands. Presumed eccrine hidrocystomas may represent cystic tumors of the eccrine sweat duct, or they may represent cystic tumors of the apocrine duct. Thus, the name hidrocystoma should be used without further specification of an eccrine or apocrine nature, unless certainty is reached by immunohistochemical characterization. Also, hidrocystomas often prove to be histologically misdiagnosed apocrine cystadenomas because of a flattened cyst wall secondary to increased intraluminal pressure.

Prediction of depressive relapse in remitted bipolar patients using corticotrophin-releasing hormone challenge test.

Abnormalities in corticotrophin (ACTH) and cortisol levels before and after corticotrophin-releasing hormone (CRH) stimulation have been reported in depressed bipolar patients. The ACTH and free cortisol response to the injection of 100 micrograms of synthetic human CRH and plasma cortisol-binding globulin (CBG) levels were measured in 42 lithium-treated patients suffering from RDC bipolar-I disorder in remission, and in 21 age- and sex-matched control subjects. A 1-year follow-up was conducted in order to assess any possible relationship between outcome and the hormonal response. Bipolar patients showed higher baseline and peak ACTH concentrations than controls. A lower net area under the ACTH concentration curve after CRH stimulation predicted depressive relapse within 6 months by multiple regression analysis. The CRH challenge test could be a potentially good predictor of depressive relapse in remitted bipolar patients.

Objective models for steroid binding sites of human globulins.

We report the application of a recently developed alignment-free 3D QSAR method [Crippen, G.M., J. Comput. Chem., 16 (1995) 486] to a benchmark-type problem. The test system involves the binding of 31 steroid compounds to two kinds of human carrier protein. The method used not only allows for arbitrary binding modes, but also avoids the problems of traditional least-squares techniques with regard to the implicit neglect of informative outlying data points. It is seen that models of considerable predictive power can be obtained even with a very vague binding site description. Underlining a systematic, but usually ignored, problem of the QSAR approach, there is not one unique type of model but, rather, an entire manifold of distinctly different models that are all compatible with the experimental information. For a given model, there is also a considerable variation in the found binding modes, illustrating the problems that are inherent in the need for 'correct' molecular alignment in conventional 3D QSAR methods.

Globulin‐Enriched Protein Supplements Shorten the Pre‐Compaction Mitotic Interval and Promote Hatching of Murine Embryos

To determine whether Synthetic Serum Substitute (SSS), which contains human globulins in addition to Human Serum Albumin (HSA), is superior to HSA alone as a protein supplement for embryo culture. Development of mouse zygotes to eight-cell/compacting morulae and to hatching/hatched blastocysts was assessed in Human Tubal Fluid (HTF) medium containing either SSS of HSA. Although there was no difference in the overall blastocyst rate at 120 h in HTF+SSS versus HTF+HSA, significantly more embryos at 54 h were at the eight-cell/compacting morula stage in HTF+SSS. At 120 h, there were more hatching/hatched blastocysts in HTF+SSS, and hatching correlated with SSS concentration. Addition of isolated globulins to HSA significantly stimulated the number of hatching/hatched blastocysts. Hatching could be "rescued" by transfer of embryos grown in HTF+HSA to globulin-containing media and prevented by removal of globulins as late as the compacted morula stage (54 h). SSS is superior to HSA alone for embryo culture. The stimulatory effects on mitosis and hatching may be mediated directly by globulins or by other components in the globulin-enriched fraction.

Immunogenicity of cholecystokinin octapeptide-conjugated antigens in pigs.

An improved antigen is required to raise antiserum titers against cholecystokinin higher than those observed in previous studies to demonstrate the effect of immunoneutralization of cholecystokinin on feed intake in swine. Four immunogenic carrier proteins were compared. Unsulfated cholecystokinin octapeptide (CCK8ns) was conjugated to each of human serum globulin (HSG), BSA, Keyhole limpet hemocyanin (KLH), and Tuberculin purified protein derivative (PPD). Forty crossbred swine (four from each of 10 litters, 78 d of age, 22 kg BW) were randomly assigned to the four conjugated antigens by litter. The primary and three booster doses of antigen were injected at 14-d intervals. A blood sample was drawn before the primary dose on d 1 to assess basal nonspecific binding of antigen. Additional blood samples were drawn on d 22, 29, 36, 43, 50, 57, 71, and 92 to follow the time course of antiserum titer expression (d 1 = day of primary dose). Titer is the serum dilution that binds 50% of 1 fmol of radiolabeled antigen. Titers, compared by ANOVA of log titer values, were different between antigens (P < .01) and between litters (P < .01). Mean titer (n = 40; 10 pigs, four samples/pig) during the period after the immune response (d 50, 57, 71, and 92) was 55, 115, 176, and 535 for BSA, HSG, KLH, and PPD, respectively. It is concluded that the carrier protein component has an important effect on immunogenicity of conjugated CCK antigens in pigs; BSA was inferior and KLH and PPD were superior to HSG.

6.5#106 - Removal of anti HLA antibody with pooled human globulin preparations

6.5#106 - Removal of anti HLA antibody with pooled human globulin preparations

New method of simultaneous and non-destructive determination of human serum albumin and globulin

A new method is described for serum albumin and serum globulin determination. This method combines the spectrophotometric method with a Kalman filter for determination of serum sample. The concentration of serum albumin and globulin is simultaneously known as soon as the absorption spectrum of the serum sample is recorded and analyzed. The operation is simple and the only manipulation is to dilute the sample with buffer solution, thus the serum sample is not destroyed. The results of the determination, compared with the bromocresol green and biuret methods, are satisfactory.

Precise localization of the human thyroxine-binding globulin gene to chromosome Xq22.2 by fluorescence in situ hybridization

The human thyroxine-binding globulin (TBG) gene has been localized to X chromosome (Xq22.2) by in situ hybridization using a biotinylated gDNA probe. This is consistent with previous mapping of the TBG gene to chromosome Xq21-q22.

Synthetic serum substitute (SSS): A globulin-enriched protein supplement for human embryo culture

The purpose of the present study was to evaluate whether an IVF protein supplement prepared from human serum albumin (HSA) and human globulins would retain performance characteristics equivalent to those reported for the commercial plasma expanders, Plasmatein (Alpha Therapeutics, Los Angeles, California) and Plasmanate (Cutter Biological, Miles Inc., Elkhart, Indiana). Pronuclear-stage human embryos were randomly divided and cultured in human tubal fluid medium (HTF) supplemented with either HSA (5 mg/mL) or Plasmatein (10%, v/v; 5 mg/ml) as a means of indirectly assessing the effect alpha- and beta-globulins have on embryonic development. Those results coupled with the known composition characteristics of Plasmatein were used as the starting basis to formulate test lots of synthetic serum substitute (SSS). Significantly (P < 0.05) more of the human embryos cultured in Plasmatein supplemented medium reached the four-cell or greater stage by 40 hr postinsemination than a comparable group cultured in HSA alone. Lot 1 of SSS, formulated with HSA (84% of total protein) and human globulins (16% of total protein) and an aqueous lipoprotein fraction derived from human plasma (Excyte IV; Miles Diagnostics, Kankakee, Illinois), produced accelerated early embryonic growth relative to control murine embryos grown in the presence of Plasmatein, however, the percentage of the embryos reaching the hatched blastocyst stage was decreased (45 vs 100%). Human embryos from seven patients, randomized to HTF medium supplemented with Plasmatein or lot 1 of SSS, showed equivalent growth at 36-40 hr postinsemination. A microprecipitate developed in media supplemented with lot 1 after several days of culture. The Excyte IV concentration was reduced and, ultimately, eliminated from the subsequent and final prototype lots of SSS. Murine embryos grown in the presence of lipoprotein free SSS showed significantly accelerated (P < 0.01) growth at 17 hr postthaw compared to Plasmatein and all embryos progressed to hatching by 41 hr. Human embryos, randomized to either Plasmatein or lot 3 of SSS, showed significantly accelerated growth (P < 0.01) when scored at 38 hr following insemination. Synthetic serum substitute provides a convient, standardized means of adding protein to media used in assisted reproductive technology (ART) procedures.

Benign myoepithelioma of the salivary glands: a true entity?

Myoepithelioma is a rare neoplasm of the salivary glands which is now recognized as an individual entity in the revised WHO classification. In this study, eleven benign tumours are presented. Most patients gave a history of a slowly enlarging mass, which was cured by surgical excision. However, one case recurred several times over 50 years, and another still has residual tumour and removal is not possible. The histological appearances included solid, myxoid and reticular growth patterns, composed predominantly of spindle shaped or plasmacytoid (hyaline) cells. Many of the tumours also contained occasional small ducts. All 11 tumours were positive for S-100 protein, variable reactions being seen for various other antigens--keratins, human milk fat globulin, carcinoembryonic antigen, alpha smooth muscle actin and vimentin. It is probable that myoepithelioma constitutes one end of a biological spectrum which also includes pleomorphic adenoma and some (non-membranous) basal cell adenomas. In practice, however, we still advocate retention of myoepithelioma as a separate diagnostic category, on the grounds that it has a range of distinctive microscopic appearances and poses its own unique problems in correct identification.

Evidence for a local role of inhibin or inhibin subunits in compensatory ovarian hypertrophy

The aim of this study was to determine whether immunoneutralization of inhibin altered compensatory ovarian hypertrophy. Crossbred postpubertal gilts actively immunized with a synthetic bovine inhibin peptide fragment (bINH) conjugated to human alpha globulins (HAG, n = 4 gilts) or HAG alone (control; n = 5) were unilaterally ovariectomized at mid-cycle. After unilateral ovariectomy, the remaining ovary was removed between day 8 and day 12 of the subsequent oestrous cycle. The number of corpora lutea per ovary was determined at each ovariectomy. Blood samples were collected at frequent intervals beginning 1 h before and continuing until the first oestrus after unilateral ovariectomy, and serum concentrations of FSH, LH, progesterone and oestradiol were determined. Inhibin antibody titres were estimated from the percentage of 125I-labelled bINH bound to serum diluted 1:4000. At unilateral ovariectomy, the number of corpora lutea per ovary was similar for bINH:HAG-immunized and control gilts (8.6 +/- 0.7 versus 7.6 +/- 0.6). During the next oestrous cycle after unilateral ovariectomy, the number of corpora lutea on each remaining ovary had doubled (P < 0.05) in controls compared with the number of corpora lutea per ovary in the previous cycle. In contrast, the number of corpora lutea remained unchanged in bINH:HAG-immunized gilts. Titre of anti-inhibin antibodies in bINH:HAG-immunized gilts was 9 +/- 1% at unilateral ovariectomy compared with 0% for controls. Alterations in serum concentrations of hormones after unilateral ovariectomy did not differ between treatment groups. Compensatory ovarian hypertrophy was blocked after unilateral ovariectomy in immunized gilts independent of alterations in serum hormones, duration of oestrous cycle, or normal ovulation rate per ovary.(ABSTRACT TRUNCATED AT 250 WORDS)