The psychosocial work environment among educators during the COVID-19 pandemic.

BACKGROUND: The education sector has been heavily impacted by COVID-19. While the impact on school-aged children has received much attention, less attention has focused on the experiences of educators. AIMS: To compare various dimensions of the psychosocial work environment and health outcomes between educators engaged in online learning to those engaged in in-person learning in the Canadian province of Ontario. METHODS: Responses from 5438 educators engaged in either online or in-person learning were collected between 23 November and 21 December 2020; three months after the start of the 2020/21 academic year in September 2020. Psychosocial outcomes included quantitative demands, work pace, predictability, role conflicts, and social support from supervisors and co-workers; assessed using an abbreviated version of the Copenhagen Psychosocial Questionnaire. Secondary outcomes included burnout and sleep troubles. Ordinary Least-Squares regression models examined adjusted mean differences in the levels of outcomes for respondents in in-person versus online learning, after adjustment for a variety of covariates. RESULTS: Compared to respondents engaged in in-person learning, respondents engaged in online learning reported less predictability, higher role conflicts and less support from supervisors and co-workers. Statistically significant differences in work pace, burnout and sleep troubles were also observed across learning modes, although these differences did not exceed previously suggested thresholds for minimum important differences. CONCLUSIONS: Important differences in the psychosocial work environment were observed between respondents engaged in in-person learning versus online learning. Addressing these differences is required, given the potential continued importance of online learning within the context of the COVID-19 pandemic and beyond.

Difference scores between single-task and dual-task gait measures are better than clinical measures for detection of fall-risk in community-dwelling older adults.

BACKGROUND: As the proportion of older adults in the population increases, so does the associated prevalence of falls, making falls the leading cause of fatal and nonfatal injuries among adults aged ≥65 years. In response, researchers and clinicians seek to develop a clinical tool that accurately predicts fall risk. These Investigations have included measures of clinical mobility and balance tests, strength, physiologically based tests, postural sway, and mean and variability of gait measures. To date, no study has concurrently explored all these measures to determine which measures, alone or in combination, emerge as the most predictive of fall risk. While there is evidence that dual-task gait conditions are sensitive indicators of fall risk, difference scores between dual-task and single-task gait conditions (DS) have not been explored. RESEARCH QUESTION: This study included outcome measures representing diverse domains (clinical mobility and balance, strength, physiological, postural sway, and mean and variability of difference scores between dual- and single-task gait conditions) to determine the combination of measures that were the most sensitive for retrospectively classifying fallers from non-fallers. METHODS: Forty-two (mean: 75.8 yrs ± 3.3) community-dwelling older adults completed a comprehensive battery of 76 measures and classified into two groups based on self-report of having one or more falls in the previous year. RESULTS: Results suggest that 11 measures captured the salient characteristics of the total cohort (fallers (N = 27) and non-fallers (N = 15) and that five gait measures were sufficient for correctly classifying fallers and non-fallers with 92.3% sensitivity and 66.7% specificity with a total model classification of 82.9%. SIGNIFICANCE: The five variables comprise mean DS of stride timing, stride width, and stride length and DS in variability for stride width and stride velocity demonstrating that difference in performance between dual-task and single-task gait trials was essential for discriminating fallers and superior to other measures.

A unique talin homologue with a villin headpiece-like domain is required for multicellular morphogenesis in Dictyostelium.

Molecules involved in the interaction between the extracellular matrix, cell membrane and cytoskeleton are of central importance in morphogenesis. Talin is a large cytoskeletal protein with a modular structure consisting of an amino-terminal membrane-interacting domain, with sequence similarities to members of the band 4.1 family, and a carboxy-terminal region containing F-actin-binding and vinculin-binding domains [1] [2]. It also interacts with the cytoplasmic tail of beta integrins which, on the external face of the membrane, bind to extracellular matrix proteins [3]. The possible roles of talin in multicellular morphogenesis in development remain largely unexplored. In Dictyostelium, a eukaryotic microorganism capable of multicellular morphogenesis, a talin homologue (TALA) has previously been identified and shown to play an important role in cell-to-substrate adhesion and maintenance of normal elastic properties of the cell [4] [5] [6]. Here, we describe a second talin homologue (TALB) that is required for multicellular morphogenesis in the development of Dictyostelium. Unlike any other talin characterised to date, it contains an additional carboxy-terminal domain homologous to the villin headpiece.

Sodium chloride enhances markedly the thermal stability of thermolysin as well as its catalytic activity.

Thermolysin, a thermophilic metalloproteinase, is markedly activated in the presence of high concentrations (1-5 M) of neutral salts. The activity increases in an exponential fashion with increasing salt concentration, and is enhanced 13-15 times with 4 M NaCl at pH 7.0 and 25 degreesC (K. Inouye, Effects of salts on thermolysin: activation of hydrolysis and synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester, and a unique change in the absorption spectrum of thermolysin, J. Biochem. 112 (1992) 335-340). In this study, the effect of NaCl on the thermal stability of thermolysin has been examined at 60-85 degreesC. The activation energy, Ea, for the thermal inactivation is 15 kcal/mol at 0 M NaCl, and increases up to 30-33 kcal/mol by the addition of 0. 5-1.5 M NaCl. Further increase in [NaCl] decreases the Ea value, and at 4 M NaCl it is almost the same as that at 0 M NaCl. Thermolysin at 0.5-1.5 M NaCl is twice as heat-stable as in the absence of NaCl. The NaCl dependence of the stability is different from that of the activity, suggesting that the effects of NaCl on activity and stability are independent. Thermolysin has been demonstrated to be not only a thermophilic enzyme but also a highly halophilic one.

Effects of the stereo-configuration of the hydroxyl group in 4-hydroxyproline on the triple-helical structures formed by homogenous peptides resembling collagen.

To explore further the recent demonstration that hydroxyproline stabilizes the triple-helical structure of collagen, two peptides containing allohydroxyproline, (aHyp-Pro-Gly)10 and (Pro-aHyp-Gly)10, were synthesized by a modified Merrifield technique which yields products of defined molecular weight. Examination of the peptides by optical rotation and circular dichroism showed that neither of them formed triple-helical structures in aqueous solution. Since the peptides had less tendency than (Pro-Hyp-Gly)10 to become helical, the results demonstrated that the trans-4-hydroxyl group of hydroxyproline makes a specific contribution to stability of the triple helix formed by (Pro-Hyp-Gly)10. Since the peptides also had less tendency than (Pro-Pro-Gly10 to become helical, the results further demonstrated that the cis-4-hydroxyl group on allohydroxyproline decreases the stability of the triple helix. The observations provided direct support for previous data indicating that incorporation of proline analogues such as allohydroxyproline into pro-alpha chains during procollagen biosynthesis prevents the polypeptides from becoming triple helical.

A voltage- and K+-dependent K+ channel from a membrane fraction enriched in contractile vacuole of Dictyostelium discoideum.

We obtained a membrane fraction enriched in the contractile vacuole by aqueous-polymer two-phase partitioning and its channel activities were analysed by incorporating it into artificial planar lipid bilayers. In asymmetrical KCl solutions (cis, 300 mM/100 mM, trans), we observed single-channel currents of a highly K(+)-selective channel with slope conductance of 102 pS and reversal potential of -20.4 mV, which corresponded to PK+/PCl- = 7. They showed bursts separated by infrequent quiescent periods. At 0 mV the mean open time was 2.0 ms. Among monovalent cations, Na+ and Li+ were impermeable, whereas Rb+ showed permeability equivalent to that of K+, although the unitary conductance was apparently reduced when the current flowed from the Rb+ containing side, suggesting that Rb+ is a permeant blocking ion. The open probability within bursts remained constant at approx.0.6 as long as the holding potential was positive on the cis side with respect to the trans side, but it decreased to 0 at negative potential. This channel was blocked by submillimolar concentrations of quinine and 30 mM TEA+. The open probability-voltage relationship showed a striking dependency on the KCl concentration on either side. This channel may play a role in water transport in this organelle.

Biphasic kinetic behavior of rat cytochrome P-4501A1-dependent monooxygenation in recombinant yeast microsomes.

Rat cytochrome P-4501A1-dependent monooxygenase activities were examined in detail using recombinant yeast microsomes containing rat cytochrome P-4501A1 and yeast NADPH-P-450 reductase. On 7-ethoxycoumarin, which is one of the most popular substrates of P-4501A1, the relationship between the initial velocity (v) and the substrate concentration ([S]) exhibited non-linear Michaelis-Menten kinetics. Hanes-Woolf plots ([S]/v vs. [S]) clearly showed a biphasic kinetic behavior. Aminopyrine N-demethylation also showed a biphasic kinetics. The regression analyses on the basis of the two-substrate binding model proposed by Korzekwa et al. (Biochemistry 37 (1998) 4137-4147) strongly suggest the presence of the two substrate-binding sites in P-4501A1 molecules for those substrates. An Arrhenius plot with high 7-ethoxycoumarin concentration showed a breakpoint at around 28 degrees C probably due to the change of the rate-limiting step of P-4501A1-dependent 7-ethoxycoumarin O-deethylation. However, the addition of 30% glycerol to the reaction mixture prevented observation of the breakpoint. The methanol used as a solvent of 7-ethoxycoumarin was found to be a non-competitive inhibitor. Based on the inhibition kinetics, the real V(max) value in the absence of methanol was calculated. These results strongly suggest that the recombinant yeast microsomal membrane containing a single P-450 isoform and yeast NADPH-P-450 reductase is quite useful for kinetic studies on P-450-dependent monooxygenation including an exact evaluation of inhibitory effects of organic solvents.

Decreased biologic activity and degradation of human [SerB24]-insulin, a second mutant insulin.

A second mutant insulin, identified as [SerB24]-insulin, has a highly hydrophilic character. To determine the biologic activity and the degradation of this mutant insulin, human [SerB24]- and [SerB25]-insulin analogues were semisynthesized from porcine insulin by an enzyme-assisted coupling method. All of the following studies on isolated rat adipocytes were performed at 37 degrees C to directly correlate the binding potency and the biologic activity. The ability of these insulins to displace 125I-porcine insulin bound to adipocytes was 0.5-2% and 1-4%, respectively, of porcine insulin. When the ability of these insulins to stimulate glucose transport and glucose oxidation was measured, both analogues had full activity at high concentrations (250 ng/ml). However, ED50 of the porcine, [SerB24]-, and [SerB25]-insulins to stimulate glucose transport was 0.37 +/- 0.05, 46.3 +/- 5.4, and 23.3 +/- 5.5 ng/ml, respectively. Similarly, for glucose oxidation, ED50 was 0.38 +/- 0.06, 33.8 +/- 3.6, and 16.6 +/- 3.4 ng/ml, respectively. Thus, the biologic activity of [SerB24]- and [SerB25]-insulins was reduced to 0.5-2% and 1-4% of that of porcine insulin, which was compatible with our previous studies under different conditions. No antagonistic effects were observed for either analogue. Degradation of 125I-labeled [SerB24]- and [SerB25]-insulins was also decreased to 62.8% and 55.8%, respectively, of 125I-porcine insulin. These results confirm the importance of the hydrophobic residues at B24 and B25 in the biologic activity of insulin; the patient having this hydrophilic insulin was considered to be in an insulinopenic state despite the hyperinsulinemia due to decreased degradation of the mutant insulin.

Molecular regulation of the hypothalamo-pituitary-adrenal axis in streptozotocin-induced diabetes: effects of insulin treatment.

Increased hypothalamo-pituitary-adrenocortical (HPA) activity in diabetes is likely important in the development of some pathologies associated with the disorder. We hypothesized that central regulation of HPA activity differs among normal, streptozotocin (STZ)-diabetic, and insulin-treated diabetic rats. Blood glucose, ACTH, and corticosterone were elevated, 8 d after inducing diabetes. Insulin treatment normalized these parameters. Plasma norepinephrine was similar in all groups, but epinephrine was lower in STZ-diabetic and higher in insulin-treated rats vs. normals. Increased ACTH with diabetes corresponded with increased hypothalamic CRH mRNA, but no change in pituitary POMC mRNA. With insulin-treatment, CRH mRNA remained elevated, and POMC mRNA was unaltered. Hippocampal MR mRNA expression was dramatically increased with diabetes and, moreover, was not normalized by insulin. No differences in GR mRNA were detected between normal and STZ-diabetic rats. However, insulin treatment increased GR mRNA levels in the paraventricular nucleus and pituitary. We postulate that, in STZ-diabetes: 1) increased HPA activity is caused by increased central drive at and/or above the level of the paraventricular nucleus and is associated with decreased epinephrine; and 2) normalized pituitary-adrenal activity with insulin may be caused by the compensatory increase in GR mRNA allowing glucocorticoid-mediated suppression of ACTH secretion despite the residual increase in central HPA activity. Thus, insulin apparently restored HPA activity at and below the pituitary but, surprisingly, not above it.

Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide.

To elucidate the ligand-receptor relationship of the natriuretic peptide system, which comprises at least three endogenous ligands, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), and three receptors, the ANP-A receptor or guanylate cyclase-A (GC-A), the ANP-B receptor or guanylate cyclase-B (GC-B), and the clearance receptor (C-receptor), we characterized the receptor preparations from human, bovine, and rat tissues and cultured cells with the aid of the binding assay, Northern blot technique, and the cGMP production method. Using these receptor preparations, we examined the binding affinities of ANP, BNP, and CNP for the C-receptor and their potencies for cGMP production via the ANP-A receptor (GC-A) and the ANP-B receptor (GC-B). These analyses revealed the presence of a marked species difference in the receptor selectivity of the natriuretic peptide family, especially among BNPs. Therefore, we investigated the receptor selectivity of the natriuretic peptide family using the homologous assay system with endogenous ligands and receptors of the same species. The rank order of binding affinity for the C-receptor was ANP greater than CNP greater than BNP in both humans and rats. The rank order of potency for cGMP production via the ANP-A receptor (GC-A) was ANP greater than or equal to BNP much greater than CNP, but that via the ANP-B receptor (GC-B) was CNP greater than ANP greater than or equal to BNP. These findings on the receptor selectivity of the natriuretic peptide family provide a new insight into the understanding of the physiological and clinical implications of the natriuretic peptide system.

No enzyme activity of 25-hydroxyvitamin D3 1alpha-hydroxylase gene product in pseudovitamin D deficiency rickets, including that with mild clinical manifestation.

Pseudovitamin D deficiency rickets (PDDR) is an autosomal recessive disorder caused by defect in the activation of vitamin D. We recently isolated 25-hydroxyvitamin D3 1alpha-hydroxylase gene and identified four homozygous inactivating missense mutations in this gene by analysis of four typical cases of PDDR. This disease shows some phenotypic variation, and it has been suspected that patients with mild phenotypes have mutations that do not totally abolish the enzyme activity. To investigate the molecular defects associated with the phenotypic variation, we analyzed six additional unrelated PDDR patients: one with mild and five with typical clinical manifestation. By sequence analysis, all six patients were proven to have mutations in both alleles. The mutations varied, and we identified four novel missense mutations, a nonsense mutation, and a splicing mutation for the first time. The patient with mild clinical symptoms was compound heterozygous for T321R and a splicing mutation. The splice site mutation caused intron retention. Enzyme activity of the T321R mutant was analyzed by overexpressing the mutant 1alpha-hydroxylase in Escherichia coli cells to detect the subtle residual enzyme activity. No residual enzyme activity was detected in T321R mutant or in the other mutants. These results indicate that all of the patients, including those of mild phenotype, are caused by 1alpha-hydroxylase gene mutations that totally abolish the enzyme activity.

A dicarba analog of beta-atrial natriuretic peptide (beta-ANP) inhibits guanosine 3',5'-cyclic monophosphate production induced by alpha-ANP in cultured rat vascular smooth muscle cells.

The synthesis and biological properties are described of [Asu7,23']-beta-ANP-(7-28) (Asu, L-alpha-aminosuberic acid), a dicarba analog of beta-atrial natriuretic peptide (beta-ANP, an antiparallel dimer of human alpha-ANP with the chains linked by 7-23' and 7'-23 disulfide bonds). This Asu-analog (referred to as analog III) displaced 125I-alpha-ANP specifically bound to cultured rat vascular smooth muscle cells (VSMC) with an apparent Ki of 2.1 x 10(-8) M, but did not stimulate formation of intracellular cGMP at 10(-8) -10(-5) M. Analog III inhibited the alpha-ANP-stimulated cGMP production in VSMC competitively with a pA2 value of 7.45 and behaved as an antagonist of alpha-ANP in rat aorta smooth muscle relaxation. In addition, beta-ANP was also shown to inhibit the alpha-ANP-induced cGMP production in a dose-dependent manner. The mechanism of action of beta-ANP is also discussed.

Involvement of B-Raf in Ras-induced Raf-1 activation.

The mechanism of Ras-induced Raf-1 activation is not fully understood. Previously, we identified a 400-kDa protein complex as a Ras-dependent Raf-1 activator. In this study, we identified B-Raf as a component of this complex. B-Raf was concentrated during the purification of the activator. Immunodepletion of B-Raf abolished the effect of the activator on Raf-1. Furthermore, B-Raf and Ras-activated Raf-1 co-operatively, when co-transfected into human embryonic kidney 293 cells. On the other hand, Ras-dependent extracellular signal-regulated kinase/mitogen-activated protein kinase kinase stimulator (a complex of B-Raf and 14-3-3) failed to activate Raf-1 in our cell-free system. These results suggest that B-Raf is an essential component of the Ras-dependent Raf-1 activator.

Isolation and sequence determination of human brain natriuretic peptide in human atrium.

We isolated human brain natriuretic peptide (human BNP) from the human atrium. Sequence analysis has revealed that it is a 32-amino-acid peptide with the sequence S-P-K-M-V-Q-G-S-G-C-F-G-R-K-M-D-R-I-S-S-S-S-G-L-G-C-K-V-L-R-R-H, which is identical to the C-terminal sequence (77-108) of the human BNP precursor deduced from the cDNA sequence. The sequence of human BNP (77-108) is preceded by Pro75-Arg76 in the human BNP precursor, which is the same processing signal as Pro97-Arg98 of the precursor of atrial natriuretic peptide (ANP). The processing of the BNP precursor occurs in the cardiocyte, although that of the ANP precursor in the cardiocyte is unclear at present.

Synthesis and biological properties of two dimeric forms of human alpha-atrial natriuretic peptide.

Two dimeric forms of human alpha-atrial natriuretic peptide (alpha-ANP) were synthesized by solution methods and compared with monomeric alpha-ANP in terms of some biological and immunochemical properties. The parallel form (beta'-ANP) and the antiparallel form (beta-ANP) were equipotent in smooth muscle relaxant activity in isolated rat aorta and their ED50 values were estimated to be 1.7 X 10(-8) M and 1.6 X 10(-8) M, respectively. Diuretic and natriuretic responses induced by beta-ANP and beta'-ANP anesthetized rats were equally less potent but exhibited a significantly longer duration than those induced by alpha-ANP. beta-ANP and beta'-ANP possessed immunoreactivities of 60-100% and 50-90% (alpha-ANP, 100% on a molar basis), respectively, with three different antisera raised against alpha-ANP-related peptides.

Preparation of F(ab')2 mu fragments from rat IgM monoclonal antibodies and their application to the enzyme immunoassay of mouse interleukin-6.

F(ab')2 fragments, herein designated as F(ab')2 mu, were prepared from rat IgM monoclonal antibodies (mAbs). The IgM was digested at a pepsin-to-IgM ratio of 1:200 (w/w) in 100 mM citrate buffer (pH 4.5) at 37 degrees C for 2 h. During digestion, the light (L) chain (27 kDa) of IgM remained undegraded, whereas the heavy (H) chain disappeared and two new bands of 44 and 48 kDa appeared. The digests were fractionated by means of hydrophobic interaction HPLC with TSKgel Phenyl-5PW. The fraction containing F(ab')2 mu was homogeneous and the recovery of antigen-binding activity was 41-52%. The molecular mass of F(ab')2 mu was estimated to be 147-153 kDa, and we concluded that the fragment was composed of two truncated H chains and two intact L chains. F(ab')2 mu was used in an enzyme immunoassay of mouse interleukin-6 and the interaction of IgM with non-specific proteins was greatly reduced, when it was converted to F(ab')2 mu fragments.

A sensitive enzyme immunoassay of human thyroid-stimulating hormone (TSH) using bispecific F(ab')2 fragments recognizing polymerized alkaline phosphatase and TSH.

Bispecific F(ab')2 fragments recognizing both human thyroid-stimulating hormone (TSH) and alkaline phosphatase (ALP) were prepared by disulfide bond exchange between F(ab')2 fragments of IgG1 monoclonal antibodies (mAbs) against TSH and ALP, and were purified to homogeneity by hydrophobic interaction HPLC. ALP was polymerized by glutaraldehyde, and a new sandwich enzyme-linked immunosorbent assay (ELISA) for TSH was developed by using the ALP polymers and bispecific F(ab')2 fragments against TSH and ALP. In this assay, the preparation of covalently linked enzyme-mAb conjugates was not needed, and the interaction of mAb with non-specific proteins was greatly reduced by the use of F(ab')2 fragments. The sensitivity for TSH was shown to increase in proportion to the degree of polymerization of ALP, and the lower detection limit obtained with the ALP trimer was 0.5 microU/ml. The sensitivity was 30 times or more higher than that of the conventional ELISA using covalently linked enzyme-mAb conjugates. The use of bispecific F(ab')2 permits the use of monomers and polymers of the signal enzyme and, thereby, regulates the sensitivity of the assay system.

Method for the preparation of bispecific F(ab')2mu fragments from mouse monoclonal antibodies of the immunoglobulin M class and characterization of the fragments.

Bispecific F(ab')2mu fragments (Bs F(ab')2mu) binding simultaneously both sialyl Lewis A antigen (SLA) and human carcinoembryonic antigen (CEA) were prepared by disulfide bond exchange between F(ab')2mu fragments derived from IgM monoclonal antibodies (mAbs) against SLA and CEA, and were purified to homogeneity in a one-step procedure of hydrophobic interaction HPLC. The final yield of Bs F(ab')2mu from F(ab')2mu fragments was 70-78%, and the purity was higher than 98%. The immunoreactivities of the Bs F(ab')2mu fragments against SLA and CEA were almost the same as those of the respective parental F(ab')2mu fragments. The dissociation constant (0.17 microM) of the Bs F(ab')2mu for CEA was in good agreement with that of the parental F(ab')2mu fragments. Although the number of applications of IgM mAbs is restricted because of the large molecular mass and low solubility, Bs F(ab')2mu might, nevertheless, be a useful tool for immunotherapy and immunodiagnosis.

The use of N-(aminobenzoyloxy) succinimide as a two-level heterobifunctional agent for the preparation of hapten-protein conjugates. Daunomycin as a model hapten with an amino group.

Three geometric ortho-, meta-, and para-isomers of N-(aminobenzoyloxy)succinimide (ABS) were synthesized, and their usefulness as a two-level heterobifunctional cross-linking agent in the preparation of hapten-protein conjugates was evaluated. The conjugation was based on the principle that ABS reacts immediately with an amino group of a hapten, and an aminobenzoyl group incorporated into the hapten is then activated by diazotization to a functional diazobenzoyl group acting on tyrosine or histidine residues of the protein. Using the anti-tumor antibiotic daunomycin (DM) as a model hapten, the three isomers of ABS were compared for their ability to conjugate DM with bovine serum albumin (BSA); DM incorporation onto a BSA molecular was found to occur to the highest degree with m-ABS, followed by p-ABS. while o-ABS completely failed to conjugate under the same coupling conditions. Using m-ABS it was possible to introduce more than 10 molecules of DM per BSA molecule. One of the DM-BSA samples was used as the immunogen for the production of anti-DM serum in a rabbit. The antibody specificity was shown to be direct to DM but not to other anti-cancer drugs (bleomycin, mitomycin C, actinomycin D and 5-fluorouracil) by the double antibody enzyme immunoassay (DEIA) using DM-beta-galactosidase conjugate as a label. An enzyme-linked immunosorbent assay (ELISA) for anti-DM IgG was developed using a DM-human serum albumin (DM-HSA) conjugate similarly prepared with m-ABS and horseradish peroxidase-conjugated goat anti-rabbit IgG as the solid-phase antigen and the labelled second antibody, respectively. This ELISA permitted us to measure accurately as little as 50 ng of anti-DM IgG per ml using a standard anti-DM IgG which had been purified from the anti-DM serum using an affinity column of Sepharose 4B with DM-HSA as the ligand. Using this ELISA as well as a sandwich enzyme immunoassay (SEIA) for total IgG, serum levels of anti-DM IgG and total IgG levels were easily monitored in a rabbit following immunization with DM-BSA. These results indicate that the use of DBS provides a novel method for preparing hapten-protein conjugates which will be useful in biochemistry and immunochemistry.

New hapten-protein conjugation method using N-(m-aminobenzoyloxy) succinimide as a two-level heterobifunctional agent: thyrotropin-releasing hormone as a model peptide without free amino or carboxyl groups.

The use of a two-level heterobifunctional agent N-(m-aminobenzoyloxy)succinimide (m-ABS) allowed us to develop a new method for preparing hapten-protein conjugates. This was demonstrated by a conjugation between thyrotropin-releasing hormone (TRH) and bovine or human serum albumin (BSA or HSA). The conjugation is based on the principle that the succinimidyl ester group of m-ABS immediately acts on an epsilon-amino group of lysine residues of carrier protein BSA (or HSA) and a m-aminobenzoyl group incorporated into the protein is then activated by diazotization to a functional m-diazobenzoyl group (m-DB) acting on a histidyl group of TRH. The TRH-BSA containing about 3.5 mol of TRH per BSA molecule, elicited the production of TRH antibody in rabbits. A new type of enzyme-linked immunosorbent assay (ELISA) for TRH was developed using the antiserum, the solid-phase antigen TRH-HSA and the commercially available horseradish peroxidase-labeled goat anti-rabbit IgG/Fab' as a marker, revealing that the ELISA was monospecific to the hormone and measured as low as 50 pg of the hormone reproducibly. Also, using the antiserum by the indirect immunoperoxidase method the distribution of immunoreactive TRH in the rat brain was demonstrated in neurons of the paraventricular nucleus and neuronal processes of the median eminence. These results strongly suggested that the use of m-ABS provided a simple and efficient new method for preparing immunogens not only for the previously reported haptens with a primary amino group(s) (J. Immunol. Methods 134 (1990) 227), but also for haptens with an imidazole, phenolic, or indole group(s) in the molecule.