The magnitude of khat use and its association with health, nutrition and socio-economic status.

Although the literature on khat (Catha edulis Forsk) is fairly extensive, and several authors have stated the potential adverse effects of habitual use of khat on mental, physical and social well-being, very few population based studies exist to substantiate those statements in Ethiopia. A house-to-house survey of a representative sample of 1200 adults from a rural Ethiopian community was conducted from January to September of 1997 to determine the prevalence of khat use and its association with health, nutritional status, mental distress, substance use, family and social functioning and economic well-being. The current prevalence of khat chewing was found to be 31.7%. Muslims more than Christians, males more than females, those between the ages 15 and 34 years more than other age groups were habitual users. The following factors were found to be significantly associated with khat use: physical illness, (OR = 1.52, 95% CI = 1.14-2.02); injuries (OR = 2.31, 95% CI = 1.42-3.79), undernutrition (OR = 1.76, 95% CI = 1.24-2.48), mental distress (OR = 8.30, 95% CI = 5.20-13.31). Family functioning among current khat users was significantly higher than non users (OR = 1.56, 95%-CI = 1.04-2.28). Social functioning and economic well-being were not significantly associated with khat use. It is concluded that a fairly large proportion of the population consumes khat and that this is related to physical and mental ill-health, although family and social functioning, and economic well-being seem to be unrelated to khat use.

Zn(2+)-selective purification of recombinant proteins from the milk of transgenic animals.

The milk of transgenic livestock is becoming a viable, large-scale source of post-translationally complex, recombinant therapeutic proteins. Recombinant vitamin K-dependent proteins such as human protein C (rhPC) and Factor IX can be produced in milk. However, rate limitations in post-translational modification such as intrachain proteolytic cleavage and gamma-carboxylation occur in the mammary gland. Thus, most desirable recombinant products often exist as sub-populations in milk because the mammary gland tends to secrete incompletely processed polypeptides. In general, a nonaffinity purification strategy by which to purify mature recombinant proteins from milk is desirable. Zn2+ is used to selectively modify ion-exchange adsorption behavior of endogenous and recombinant milk proteins through conformational changes which cause aggregation and or precipitation. Zn(2+)-selective precipitation of milk and recombinant proteins results in the purification of active rhPC at high yield from the milk of transgenic pigs using expanded bed chromatography. This method selects for rhPC which is both heterodimeric and properly gamma-carboxylated. Due to the homology of milk proteins among different species, this same Zn(2+)-selective precipitation strategy is useful for developing purification methods for other recombinant proteins from the milk of transgenic livestock.

Transanal excision of rectal carcinoma.

Twenty-two men and 16 women with a mean age of 67 years were treated for rectal carcinoma by transanal excision. Patients presented with rectal bleeding (63%), change in bowel habits (11%), rectal pain (4%), or were asymptomatic and discovered on screening proctosigmoidoscopy (22%). The tumors were located from the anal verge to 8 cm proximally and ranged in size from 1 to 4 cm. Pathologic findings included adenocarcinoma (92%), squamous cell carcinoma (4%), and cloacogenic carcinoma (4%). Postoperative hospitalization averaged two days (0 to 29 days). One patient died of a perioperative myocardial infarction for an operative mortality of 3 per cent. Morbidity was 7 per cent and included urinary retention and pneumonia. Postoperative radiation therapy was administered to 11 patients with either undifferentiated tumors or invasion into the muscularis propria. Follow-up in these 38 patients averaged 30 months. One patient died of metastatic carcinoma, and two patients developed local recurrence that was treated successfully by a low anterior resection or abdominoperineal resection. Transanal excision of rectal carcinoma can be performed in properly selected patients with good overall survival and local control.

Purification of recombinant human granulocyte-macrophage colony-stimulating factor from the inclusion bodies produced by transformed Escherichia coli cells.

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), produced as inclusion bodies in genetically transformed Escherichia coli cells was purified to homogeneity by a three-step chromatographic procedure involving hydrophobic interaction, ion exchange and gel filtration. Each purification step is reproducible and well suited for process-scale operations. The purification process also leads to a significant decrease in DNA and endotoxin levels in the final product. Of the three gel media used, Phenyl Sepharose 6 FF (high sub) was most effective in reducing the DNA content (by a factor of ca. 2000) while Superdex 75 prep grade was more effective for removing endotoxins (reduction factor ca. 15). The recovery of purified rhGM-CSF was 35% by enzyme-linked immunosorbent assay and 70% by a biological assay method. The overall purification factor obtained was about 4.6, which is in the range of those reported for recombinant proteins produced in E. coli as inclusion bodies. The purified rhGM-CSF is an acidic protein (pI = 5.4) and has a specific activity of ca. 3.3 x 10(7) units/mg, which is in excellent agreement with that reported for its natural counterpart. Its monomer molecular mass of 14,605, as determined by electrospray mass spectrometry, corresponds exactly to the mass calculated from its cDNA sequence. Its amino acid composition and partial NH2-terminal sequence (up to seventeen residues) are also identical with those reported for this protein. These and other results confirm the identity of the purified rhGM-CSF with its natural counterpart. However, the results also showed that it is apparently heterogeneous from its NH2-terminal side as it is composed of three polypeptides having Met, Ala and Pro as the NH2-terminal residues in which the intact Met analogue accounts for 60% for the mixture. This heterogeneity does not seem to have any biological significance since the specific activity of the purified rhGM-CSF is identical with that of its natural counterpart.

Production, purification and characterization of recombinant human interferon gamma.

An essentially three-step chromatographic purification procedure, i.e., ion-exchange, immobilized metal ion affinity and size-exclusion chromatography, is described for the purification to homogeneity of recombinant human interferon-gamma (rhIFN-gamma) from the inclusion bodies produced in genetically transformed Escherichia coli cells. Batchwise adsorption of the cloudy solution of renatured rhIFN-gamma obviated the need for high-speed centrifugation to clarify the suspension. This step effectively removed about 70% of extraneous protein impurities. The established purification process is reproducible and leads to a total recovery of 32%. Pilot-scale processing of E. coli cells grown in a 30-l fermentor gave about 70 mg of a homogeneous preparation of rhIFN-gamma. The specific biological activity of purified rhIFN-gamma is ca. 3.4 x 10(7) I.U./mg protein, which is comparable to that of its natural counterpart. It is basic protein (pI greater than pH 9) with a monomer relative molecular mass of 15,000. It behaves, however, as a dimer on size-exclusion chromatography. Its partial NH2-terminal sequence is identical with that established for the rhIFN-gamma. However, its amino acid composition and its relative molecular mass (15,067 as determined by electrospray mass spectrometry) indicate that the purified protein is a truncated form lacking fifteen amino acid residues from its carboxyl-terminal side. This modification does not seem to have any adverse effect on its biological potency. The levels of DNA, bacterial endotoxins and Ni(II) ions in the final product were determined.

Immobilized metal ion affinity chromatography. Effect of solute structure, ligand density and salt concentration on the retention of peptides.

The adsorption characteristics of a variety of synthetic peptide hormones and di-, tri- and tetrapeptides on Cu(II) immobilized on two commercially available high-performance chelating gels run under various experimental conditions are described. Methods for determining the concentration of immobilized Cu(II) in situ are also described. The Cu(II)-charged columns exhibit a net negative charge as judged from the significantly higher retention of some basic peptides in the absence of NaCl in the equilibration and elution buffers. At higher NaCl concentrations (2-4 M), aromatic interactions seem to be superimposed on the metal ion affinity characteristics of the peptides. The relationship between resolution of peptides and the concentration of immobilized Cu(II) ions has also been established for the Chelating Superose gel where 40 mumol Cu(II) ml-1 gel apparently gives the optimum resolution. The nature of the gel matrix also plays a role in the resolution of some peptides, the extent of which is difficult to predict. The results obtained also suggest that peptides containing aromatic and hydroxy amino acids are retarded more than those which lack them. Moreover, these same amino acids apparently strengthen the existing strong binding of peptides containing His, Trp or Cys to a Chelating Superose-Cu(II) column. Dipeptides with C-terminal His (i.e., X-His) are neither bound nor retarded on a column of Chelating Superose-Cu(II) whereas those having the structure His-X are strongly bound. Some tri- and tetrapeptides containing His were also found not to bind to the column. The underlying cause of this anomalous adsorption behaviour is discussed and is ascribed to "metal ion transfer" arising from the relatively higher affinity of such peptides towards immobilized Cu(II) ions than the chelator groups (iminodiacetate) which are covalently bound to the gel matrix.

Separation and enrichment of alkylated globin chains as a means of improving the sensitivity of hemoglobin adduct measurements.

Measurement of adducts of hemoglobin is a reliable and quantitative method for monitoring exposure to genotoxic chemicals. To make the approach applicable to the low levels of adducts originating from exposure to chemicals in the environment, increased sensitivity of the analytical procedures is required. The method presented here is based on quantitative determination of low levels of adducts after purification and enrichment of chemically modified (adducted) globin chains on CM-Sepharose CL-6B. In the developmental work, human globin was used after alkylation by radiolabelled ethylene oxide, styrene oxide or N-ethyl-N-nitrosourea. Ethylene oxide reacts mainly with the amino terminal valine and nitrogens in the imidazole ring of histidine, while N-ethyl-N-nitrosourea has a particularly high reactivity towards free carboxy groups of acidic amino acids. Globin chains with adducts to the carboxy groups were especially easy to separate from the non-modified chains. Ethyl adducts to carboxy groups in hemoglobin were shown to be sufficiently stable in vivo to be used for dose monitoring.

High-performance immobilized metal ion affinity chromatography of peptides: analytical separation of biologically active synthetic peptides.

The separation of more than 30 biologically active synthetic peptides and their analogs on a high-performance immobilized metal ion affinity chromatography column is described. The metal chelating gel (TSK gel chelate-5PW) contains iminodiacetic acid (IDA) covalently coupled to a hydrophilic, resin-based matrix with a bead diameter of 10 micron. The retention of the peptides on Cu(II), Ni(II), and Zn(II) ions immobilized on the chelating gel showed that some of them can be separated by isocratic elution while the majority of them are retained and are separated into distinct fractions by elution with a linear imidazole gradient or with a continuously decreasing pH gradient. Of the three immobilized metal ions investigated here, the IDA-Cu(II) chelate column gave the best resolution irrespective of the type of gradient used. This is amply illustrated by the resolution of angiotensins I and II and their seven synthetic analogs. The results obtained here serve as guidelines for the future exploitation of this separation method for the efficient fractionation of a wide variety of peptides on an analytical or preparative scale.

A one-step purification method for monoclonal antibodies based on salt-promoted adsorption chromatography on a 'thiophilic' adsorbent.

A convenient and fast method for the purification of mouse monoclonal antibodies from the culture media of cloned cells or from ascites fluids by means of salt-promoted chromatography on a 'thiophilic' adsorbent is described. The adsorbent has a capacity to adsorb about 20 mg/ml of immunoglobulins and a broad specificity towards immunoglobulins derived from various animal species irrespective of the type or subclass to which they belong. The recovery of the purified IgG is better than 90% while that for IgM is considerably less, probably due to dissociation occurring during the adsorption-desorption process. This one-step procedure also leads to a considerable concentration of dilute solutions of immunoglobulins. Moreover, the purified Igs are eluted by an essentially salt-free buffer at near neutral pH thus obviating the need for post-treatment of the sample before storage or subsequent conjugation to enzymes for use in immunoassays. This purification method is also well suited for large-scale operations since sample preparation requires only the addition of 0.5 M K2SO4 to the ascited fluid or cell culture medium. The degree of purification obtained is, in certain instances, comparable to that obtained by biospecific affinity chromatography based on antigen-antibody interactions. In contrast to immunosorption and desorption methods, however, there is no risk of contamination of the immunoglobulins purified on the 'thiophilic' adsorbent by foreign proteins.

High-performance analytical applications of immobilized metal ion affinity chromatography.

The separation of three sets of standard protein mixtures on a high-performance immobilized metal ion affinity chromatography (HP-IMAC) column by elution with linear gradients of imidazole is described. The affinity of the test proteins for the immobilized metal ions follows the order Cu2+ greater than Ni2+ greater than Zn2+. The iminodiacetic acid-Cu2+ column gives the best resolution of all three protein mixtures and is the only immobilized metal ion column that can be used for elution of absorbed proteins with a decreasing pH gradient. An application of HP-IMAC for the separation of monoclonal IgG from mouse ascites fluid is also outlined. This versatile separation method is thus suitable for both analytical and preparative separations of proteins and peptides resulting in high recoveries and good reproducibility. The leakage of immobilized metal ions from the TSK gel chelate-5PW is apparent if the column is eluted by buffers containing low concentrations of (i) glycine or (ii) primary amines at round neutral pH. Considerable amounts of immobilized Zn2+ and Ni2+ ions also leak from the column by washing with buffers of pH 4.5 or lower. However, all three immobilized metal ions are stable toward exposure to low concentrations of imidazole (up to 50 mM) in phosphate buffers between pH 6.5 and 8.0. Adsorbed proteins could thus be eluted conveniently by using linear gradients of imidazole to give reproducible results. Moreover, this elution procedure made it possible to use the IMAC columns for repeated runs without the need for regeneration and recharging of the columns with fresh metal ions after each use.

Interaction of proteins with immobilized Cu2+. Quantitation of adsorption capacity, adsorption isotherms and equilibrium constants by frontal analysis.

The interaction of lysozyme, ovalbumin, bovine and pig serum albumins with Cu2+ immobilized on Chelating Sepharose Fast Flow or TSK gel chelate-5PW was studied by frontal analysis at various initial concentrations of these solutes. The chromatographic data so obtained served as a basis for evaluating some relevant affinity chromatography parameters by adapting previously reported equations to this system. The TSK-based adsorbent had lower adsorption capacity for all the model proteins compared to the agarose-based adsorbent, due primarily to its lower porosity which has a marked influence on the accessibility of the immobilized ligand to the proteins. On the other hand, the TSK-based adsorbent offers almost ideal conditions for studying adsorption equilibria under column chromatographic conditions. The adsorption capacity of these adsorbents for the model proteins ranges from about 0.6 to 7 mumol/ml, equivalent to 40-100 mg/ml, of adsorbent. The following equilibrium constants for the interaction of the proteins with immobilized Cu2+ were obtained: lysozyme, 1.8.10(4); ovalbumin, 1.5.0(5); BSA, 1.7.10(5); PSA, 3.7.10(5) and imidazole, 8.10(3) M-1. Despite the comparatively low affinity of imidazole for the adsorbent, it is an effective competing ligand, at comparatively high concentrations, for adsorbed proteins primarily because all adsorption sites are available to it. The results obtained suggest that about 1/3 to 1/2 of the potential adsorption sites on the model proteins are involved in forming coordination complexes with Cu2+ immobilized to covalently bound iminodiacetate groups on insoluble gel matrices.

Chick embryo nerve growth factor. Fractionation and biological activity.

A nerve growth factor (NGF)-like factor initiating nerve fibre outgrowth from sympathetic ganglia in culture was partially purified from chick embryo extract by cation-exchange chromatography followed by hydrophobic interaction chromatography on octylsulfide agarose. The NGF-like factor was markedly activated upon gel filtration in the presence of 6 M urea. Further analysis of the activated chick NGF by immunoblotting following SDS-PAGE, and by inhibition of bioassay response using antibodies to mouse beta NGF demonstrated a distinct antigenic cross-reactivity. The size of the chick embryo NGF was also indistinguishable from that of the mouse beta NGF with a molecular weight (MW) of about 14,000. The findings demonstrate directly the presence of biologically active NGF protein in the developing 18-day chick embryo.

Epidermal growth factor from deer (Cervus elaphus) submaxillary gland and velvet antler.

Epidermal growth factor (EGF)-like activity was isolated for the first time from the submaxillary gland (SMG) and the velvet antler of red deer (Cervus elaphus) by a combination of Sephadex gel or DEAE-Sephacel and IMAC columns in succession. The semipurified cervine EGF-like activity (cEGF), with specific activity of 4.7 ng/micrograms protein from the velvet tissues, can generate a completely parallel competitive binding curve against mouse EGF in both radioreceptor assay (RRA) and radioimmunoassay (RIA). Mitogenic activity of EGF from both tissues was demonstrated by stimulating the incorporation of [3H]thymidine in two different cell lines of fibroblast culture in a dose-dependent manner. The velvet layer may be the site of EGF synthesis outside the SMG.

The effect of various coupling methods on the adsorption of serum proteins by immobilized concanavalin A.

Concanavalin A was coupled to Sepharose 6B after activation by cyanogen bromide, divinyl sulfone, or glutaraldehyde and its adsorption behavior toward human serum proteins was investigated. The capacity and selectivity of the lectin were influenced markedly by the method used for its immobilization. When coupled to agarose via CNBr, the resulting absorbent showed the highest capacity and the lowest selectivity relative to the other two derivatives. When coupled to agarose via divinyl sulfone, the lectin exhibited high selectivity but its adsorption capacity was significantly reduced. Coupling to agarose via glutaraldehyde gave an absorbent that behaved, in some respects, differently from the other two. The variability in the adsorption behavior of the immobilized concanavalin A is attributed in part to variations in the degree of multipoint attachment of the lectin or its subunits to the agarose matrix. The selectivity increases also with increasing sample load, irrespective of the coupling method used, apparently due to protein-protein displacement.

A high-capacity hydrophobic adsorbent for human serum albumin.

A simple method, based on salting out hydrophobic interaction chromatography, for the efficient removal of trace amounts of serum albumin from partially purified protein preparations is described. The method is also successfully applied for the purification of albumin from Cohn fraction IV, a by-product obtained from the commercial fractionation of human serum proteins by the ethanol precipitation procedure. About 70% of the adsorbed albumin can be eluted by buffer of low ionic strength and can thus be lyophilized directly, if required. The adsorbent can be used for several cycles of adsorption and desorption without affecting its selectivity or capacity. Its adsorption properties and capacity for serum albumin are compared with those of the commercially available adsorbent Blue Sepharose CL-6B.

Isolation of the epidermal growth factor from the shrew submaxillary gland.

The epidermal growth factor (EGF) from the submaxillary glands of the Chinese Shrew (Suncus murinus) is purified to apparent homogeneity by using a sequence of four chromatographic steps, viz. gel filtration on Sephacryl S-200, affinity chromatography on immobilized Ni, hydrophobic interaction on phenyl-Sepharose CL-4B and reverse-phase HPLC. An 800-fold increase in specific activity and an overall recovery of 46% were achieved. The most effective step in its purification is the successful use of immobilized metal ion affinity chromatography (IMAC). This method was very selective, reproducible and requires a minimum of sample pre-treatment prior to chromatography.

Thiophilic adsorption--a new method for protein fractionation.

Divinylsulphone-activated agarose to which mercaptoethanol is coupled showed very selective group adsorption of human serum proteins, in particular the immunoglobulins. The adsorption increases markedly in the presence of high concentrations of neutral water-structure forming salts and is distinct from adsorptions based on hydrophobic interaction. A characteristic feature of this new type of adsorbent is the structure of the groups attached to the polymer, P, i.e., R-S-CH2-CH2-SO2-CH2-CH2-O-P, where R is a small aliphatic residue. Our results indicate that the thioether sulphur and the adjacent sulphone group act cooperatively and are apparently necessary to maintain the distinct behaviour of such absorbents.

Synthesis of new hydrophobic adsorbents based on homologous series of uncharged alkyl sulphide agarose derivatives.

A homologous series of uncharged thioalkyl derivatives of agarose were prepared by a simplified synthetic route and their adsorption behaviour towards human serum proteins was evaluated and compared with that of a commercially available alkyl ether derivative of agarose. The influence of the spacer arm length on the adsorption efficiency was also investigated. The degree of substitution of the derivatives can be estimated conveniently by sulphur analysis. The four different types of thiolkyl derivatives (C6, C8, C12 and C14) investigated here behave in all respects like hydrophobic adsorbents. The coupling yield obtained is high (75% or more) and is better than that obtained by alternative synthetic routes reported so far. The adsorption capacity towards serum proteins of the various derivatives increases with increasing alkyl chain length and degree of substitution. Desorption is achieved by a progressive decrease in the polarity of the eluent and the recovery of the applied material is in the range 80-90%. The role played by the thioether as a possible modulator of the observed hydrophobic adsorption is discussed. For the group separation of serum proteins the optimum adsorbent, as regards capacity combined with ease of elution of adsorbed material, should be substituted with chains of six or eight carbon atoms and have a ligand concentration in the range 80-120 mumole g-1 dry gel.

The labelling of an axonal membrane component with 4-(N-maleimido) benzyltrimethylammonium, a reagent capable of affinity-labelling the alpha-subunit of the nicotinic acetylcholine receptor.

Membrane vesicles, isolated from crustacean axons, were treated, following disulfide reduction, with 3H-NEM or with 3H-MBTA. SDS polyacrylamide gel electrophoresis showed that exposure to NEM (a nonspecific thiol reagent) resulted in the labelling of several peptide bands, while with MBTA only a single band with a molecular weight of 50,000 was labelled. Reaction with MBTA (believed to be a specific label for the nicotinic acetylcholine receptor) could be largely prevented by preincubation with d-tubocurarine or bromacetylcholine.