Induction of experimental allergic neuritis in the BN rat: P2 protein-specific T cells overcome resistance to actively induced disease

T lymphocyte lines specific for the peripheral nerve myelin protein P2 were selected from the lymph nodes of Brown Norway (BN) rats immunized with bovine P2 protein in complete Freund's adjuvant. These T cells expressed the W3/25+, OX8-phenotype and responded specifically to bovine P2 protein, but not to PPD or bovine basic protein, in T cell proliferation assays. When injected i.v. into syngeneic recipients, BN P2-specific T cell lines induced both clinical and histologic signs of experimental allergic neuritis (EAN), overcoming the resistance of this rat strain to actively induced EAN. Although the histopathology of the disease was indistinguishable from that seen in T cell-mediated EAN in the Lewis rat, disease onset was considerably later, 7 to 8 days after cell transfer, as opposed to 4 days in Lewis. This lag phase between inoculation and disease onset could not be further reduced even by raising the cell dose to 50 X 10(6) cells/host. The fine specificity of the T cell response to P2 differs between Lewis- and BN-derived T cell lines. At least one neuritogenic epitope for each strain was present in the cyanogen bromide-derived peptide CB2 (residues 21-113), as shown by the ability of CB2-specific T cell lines derived from each strain to transfer EAN to the appropriate host strain. However, neuritogenic BN T lines fail to mount a response to the sequence 53-78 (SP4), which encompasses an epitope that is neuritogenic for Lewis rats. These results demonstrate that the resistance of BN rats to actively induced EAN is not due to the lack of appropriate P2-specific autoreactive T cell clones in the normal T repertoire. Furthermore, the results suggest that two distinct epitopes of P2 are responsible for EAN in Lewis and BN rats.     

Conformation of Bovine Nerve Root P2 Protein: Characteristics of the Molecule from Circular Dichroism Spectra

Abstract: Circular dichroism (CD) was used to study the conformations of bovine nerve root P₂ basic protein, its reduced and carboxymethylated derivative (RCM-P₂), and its large cyanogen bromide fragment (CN1). Data in the far UV show that both the parent protein and RCM-P₂ have conformations dominated by a large amount of β structure. However, the CN1 peptide appears to exist in a largely unordered conformation. Since CN1 lacks short (20 residue) amino- and carboxy-terminal segments of the P₂ protein, the spectral data suggest that these regions are important for determining and/or maintaining folding of the P₂ protein in aqueous solutions. The P₂ protein was found to have a distinctive CD spectrum in the near UV. The characteristics of molar ellipticities indicate that the spectrum contains significant contributions from tyrosine residues, and that these contributions suggest different environments for the two tyrosines in P₂ protein. Both environments depend on protein conformation, since CD side chain absorptions are lost when P₂ protein is denatured with 5 M urea.     

A permanent rat T cell line that mediates experimental allergic neuritis in the Lewis rat in vivo

A rat T cell line of the "helper" phenotype (W3/25-positive, OX 8-negative) has been derived from Lewis rats inoculated with P2 protein isolated from bovine PNS myelin. The line LiP2/A is exquisitely specific for P2 protein, exhibiting no reactivity to bovine basic protein or to PPD. In addition to responding strongly to the intact P2 protein, the line cells show some response to a synthetic peptide containing the neuritogenic amino acid sequence of P2 protein (SP-B, residues 66-78). Intravenous inoculation of naive rats with as few as 10(4) activated LiP2/A cells leads to the onset of mild clinical signs of experimental allergic neuritis. Higher doses of cells lead to more severe clinical disease. Histologic examination of clinically ill animals confirmed the disease as EAN. The pathologic lesions were confined to the PNS and spared the central nervous system. The lesions consisted of marked perivascular cuffs and infiltrates of inflammatory cells associated with marked degenerative changes--demyelination and some axonal degeneration.     

Identification of the Palmitoylation Site in Rat Myelin P0 Glycoprotein

Abstract: P₀ glycoprotein, the major protein of PNS myelin, contains approximately 1 mol of covalently bound long-chain fatty acids. To determine the chemical nature of the fatty acid-protein linkage, P₀ was labeled in rat sciatic nerve slices with [³H]palmitic acid and subsequently treated with various reagents. The protein-bound palmi-tate was released by incubation with the reducing agents dithiothreitol and 2-mercaptoethanol, and with 1 M hydrox-ylamine at pH 7.5. In addition, P₀ was deacylated by treatment with 10 m M NaBH₄ with the concomitant production of [³H]hexadecanol, indicating that the fatty acid is bound in a thioester linkage. This conclusion was supported further by the fact that deacylation with hydroxylamine generated free thiol groups, which were titrated with [¹⁴C]-iodoacetamide. To identify the cysteine residue involved in the thioester linkage, [¹⁴C]carboxyamidomethylated P₀was digested with trypsin and the resulting peptides analyzed by reversed-phase HPLC. Identification of the radioactive protein fragments by amino acid analysis and amino-terminal peptide sequencing revealed that Cys¹⁵³ in rat P₀ glycoprotein is the acylation site. The acylated cysteine is located at the junction of the putative transmem-brane and cytoplasmic domains. This residue is also present in the P₀ glycoprotein of other species, including human, bovine, mice, and chicken.     

Bovine Peripheral Nervous System Myelin P2 Protein: Chemical and Immunological Characterization of the Cyanogen Bromide Peptides

Cleavage of bovine P2 protein by cyanogen bromide (CNBr) produced peptide fractions CN1, CN2, and CN3 which were isolated by gel filtration chromatography. CN2 was found to contain two NH2-terminals (lysine and valine) and accounted for both of the cysteine residues of P2. When reduced carboxymethylated P2 (RCM-P2) was digested with CNBr, peptides CN1 and CN3 were obtained as were (1) a peptide with NH2-terminal lysine (Lys) that contained no homoserine and only one cysteine residue and (2) a peptide with NH2-terminal valine (Val) that was co-eluted with CN3. These data and the chemical characterization of all the CNBr peptides obtained from P2 and RCM-P2 suggest that isolated P2 protein has a structure composed of the CNBr peptides in the order CN3-CN1-CN2(Val)-CN2(Lys) with an intrachain disulfide bond between the cysteine residues located in the two constituent peptides of CN2, CN2(Lys) and CN2(Val). To locate the neuritogenic region(s) within the P2 protein structure, CN1, CN2, and CN3 were tested for the ability to induced experimental allergic neuritis (EAN) in Lewis rats. The disease-inducing sites of P2 protein were found only in CN1; neither CN2 nor CN3 produced disease. EAN induced by CN1 was comparable to that induced with P2 protein as determined by disease onset, clinical symptoms, and histologic lesions.     

PROTEIN COMPOSITION OF MYELIN OF THE PERIPHERAL NERVOUS SYSTEM

Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P₀), two uncharacterized proteins, and two basic proteins (P₁ and P₂). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P₁ and P₂ proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P₂ protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P₀, P₁ and P₂ proteins from rabbit sciatic nerve and P₀ and P₂ proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P₁ protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P₀ and P₂ proteins are unique to the PNS.     

Prevention and therapy of experimental autoimmune neuritis by an antibody against T cell receptors-alpha/beta.

The mAb R73 directed to the TCR-alpha/beta of rat lymphocytes was tested for its therapeutic potential during the effector phase of experimental autoimmune neuritis (EAN) in Lewis rats. EAN can be actively induced by immunization with bovine peripheral nerve myelin, bovine P2 protein, or a peptide containing its neuritogenic epitope and serves as a model of the human Guilain-Barré syndrome. Adoptive transfer of activated P2-specific T lymphocytes also produces the monophasic disease (AT-EAN) characterized by inflammation and demyelination of peripheral nerves and highlights the central role of T lymphocytes in the pathogenesis of EAN. A single administration of the mAb R73 immediately after injection of activated P2-specific T line cells completely prevented the development of clinical and electrophysiologic signs of EAN in most animals and greatly alleviated the disease in the others. In further experiments mAb R73 was applied after the appearance of first clinical signs of EAN actively induced by immunization with a neuritogenic peptide or bovine peripheral nerve myelin. In both cases the anti-TCR-alpha/beta mAb reversed clinical signs of EAN and prevented the development of peripheral nerve dysfunction. In vivo and in vitro data suggest that impairment of Ag recognition and T cell function by occupancy of the TCR and R73-induced TCR-modulation rather than depletion of TCR-alpha/beta-bearing lymphocytes is the decisive mechanism underlying suppression of EAN that is apparent already within 48 h of the first R73 injection.     

Ultrastructural Localization of P2 Protein in Actively Myelinating Rat Schwann Cells

Abstract: The myelin specific protein, P₂, was localized immunocytochemically in electron micrographs of 4-day-old rat peripheral nerve by a preembedding technique. P₂ staining was restricted to Schwann cells that had established a one-to-one relationship with an axon. P₂ antiserum produced a diffuse staining throughout the entire cytosol of myelinating Schwann cells. In addition, the cytoplasmic side of Schwann cell plasma membranes and the membranes of cytoplasmic organelles that were exposed to cytosol were stained by P₂ antiserum. This cytoplasmic localization of P₂ protein is similar to that described for soluble or peripheral membrane proteins that are synthesized on free ribosomes. P₂ antiserum stained the cytoplasmic side of Schwann cell membranes that formed single or multiple loose myelin spirals around an axon. In the region of the outer mesaxon, P₂ antiserum stained the major dense line of compact myelin. These results demonstrate that P₂ protein is located on the cytoplasmic side of compact myelin membranes and are consistent with biochemical studies demonstrating P₂ to be a peripheral membrane protein.     

Glycoprotein Biosynthesis in Peripheral Nervous System Myelin: Effect of Tunicamycin

Abstract: The effect of an inhibitor of N -glycosylation of glycoproteins, tunicamycin, on synthesis of PNS myelin proteins was investigated in vitro by using chopped sciatic nerves or spinal roots of 21-day-old Wistar rats. Tunicamycin when incubated with these nerves in the presence of ³H-labeled fucose, mannose, or glucosamine inhibited the uptake of radioactivity into myelin proteins including some high-molecular-weight proteins, P₀, 23K protein, and 19K protein by amounts ranging from 42 to 79%. Uptake of ¹⁴Camino acid mixture was inhibited much less by tunicamycin, but a new radioactive protein peak appeared when the protein mixtures had been separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This protein ran directly in front of the P₀ peak, did not correspond to any bands stained by Fast green, and was not labeled by fucose. This peak appeared in increasing larger proportions with progressive time of incubation of nerves with ³H amino acids in the presence of tunicamycin. The new protein, which cross-reacts with P₀ antiserum, was tentatively identified as a nonglycosylated P₀ protein that appears to be almost as well incorporated as P₀ into the subcellular fraction containing myelin. At this time it is not possible to determine whether the unglycosylated P₀ is actually assembled into a site and configuration like that of P₀.     

Irreversible blockade of sigma-1 receptors by haloperidol and its metabolites in guinea pig brain and SH-SY5Y human neuroblastoma cells

We evaluated the effect of haloperidol (HP) and its metabolites on [³H](+)-pentazocine binding to σ₁ receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P₁, P₂ and P₃ subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other σ₁ antagonists or (−)-sulpiride), [³H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of σ₁ receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-α-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked σ₁ receptors in guinea pig brain homogenate and P₂ fraction in vitro . We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated σ₁ receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P₂ fraction membranes, which suggests that HP is metabolized to inactivate σ₁ receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of σ₁ receptors in brain homogenates. These results suggest that HP may irreversibly inactivate σ₁ receptors in guinea pig and human cells, probably after metabolism to reduced HP.     

Peptide 53-78 of myelin P2 protein is a T cell epitope for the induction of experimental autoimmune neuritis.

We have recently described the clinical and pathological features of experimental autoimmune neuritis (EAN) in Lewis rats inoculated with varying doses of a synthetic peptide corresponding to the amino acid residues 53-78 of bovine P2 protein (SP-26). Immunization with this synthetic peptide was able to induce severe clinical and pathological characteristics of EAN. We are now reporting that, SP-26 T cell lines derived from spleen and lymph node cell populations of such immunized rats, upon being triggered by SP-26, can adoptively transfer severe clinical and histological signs of EAN to naive syngeneic recipients. The disease appears 7-8 days postinoculation of the cells and persist 5-10 days. The pathological features were indistinguishable from SP-26-induced active EAN which appears 12-15 days after sensitization. Examination of the surface phenotype of the cells that were used for the passive transfer of EAN by FACS analysis, showed majority of the cells to be CD4+, Ia+ cells.     

Functional Modulation of P2×2 Receptors by Cyclic AMP-Dependent Protein Kinase

Abstract: It is generally believed that protein phosphorylation is an important mechanism through which the functions of voltage- and ligand-gated channels are modulated. The intracellular carboxyl terminus of P_2×2 receptor contains several consensus phosphorylation sites for cyclic AMP (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC), suggesting that the function of the P_2×2 purinoceptor could be regulated by the protein phosphorylation. Whole-cell voltage-clamp recording was used to record ATP-evoked cationic currents from human embryonic kidney (HEK) 293 cells stably transfected with the cDNA encoding the rat P_2×2 receptor. Dialyzing HEK 293 cells with phorbol 12-myristate 13-acetate, a PKC activator, failed to affect the amplitude and kinetics of the ATP-induced cationic current. The role of PKA phosphorylation in modulating the function of the P_2×2 receptor was investigated by internally perfusing HEK 293 cells with 8-bromo-cAMP or the purified catalytic subunit of PKA. Both 8-bromo-cAMP and PKA catalytic subunit caused a reduction in the magnitude of the ATP-activated current without affecting the inactivation kinetics and the value of reversal potential. Site-directed mutagenesis was also performed to replace the intracellular PKA consensus phosphorylation site (Ser⁴³¹) with a cysteine residue. In HEK 293 cells expressing (S431C) mutant P_2×2 receptors, intracellular perfusion of 8-bromo-cAMP or purified PKA catalytic subunit did not affect the amplitude of the ATP-evoked current. These results suggest that as with other ligand-gated ion channels, protein phosphorylation by PKA could play an important role in regulating the function of the P_2×2 receptor and ATP-mediated physiological effects in the nervous system.     

Basic proteins of rodent peripheral nerve myelin: immunochemical identification of the 21.5K, 18.5K, 17K, 14K, and P2 proteins

Abstract: Proteins in peripheral nervous system and central nervous system myelin and homogenates of sciatic nerve and brain from young and adult mice and rats were characterized with affinity-purified anti-P₂ and anti-myelin basic protein sera after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Using this method we have identified a component of rodent peripheral nervous system myelin as P₂ protein. Peripheral nervous system myelin also showed the presence of four basic proteins in addition to P₂ protein. These were found to be analogous to the 14, 17, 18.5, and 21.5K species found in the central nervous system myelin. A number of high-molecular-weight proteins were also detected with anti-myelin basic protein serum in peripheral nervous system, as well as central nervous system myelin. In addition, we report the presence of a high-molecular-weight P₂ cross-reactive protein in rodent brain stem homogenates, but not in central nervous system myelin.     

Changes of inositol phosphates during decapitation-induced lateral bud break of Malus domestica

An increase in phosphatidylcholine (PC), phosphatidyl-ethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI) occurred during bud break induced by decapitation. Inositol-1-phosphate [Ins(l)P₁], inositol-1,4-bisphosphate [Ins(1,4)P₂], and inositol-1,4,5-triphosphate [Ins(1,4,5)P₃] were found in apple buds and increased progressively following decapitation. Ins(1)P₁ and Ins(1,4)P₂ peaked 48 h after decapitation and Ins(1,4,5)P₃ peaked 72 h after decapitation during the metabolic transition when buds emerged from dormancy. Ins(1,4)P₂ and Ins(1,4,5)P₃ levels declined there after. The lateral buds on shoots with intact terminals and decapitated shoots treated with indole-3-acetic acid (IAA) in the terminals tip remained dormant and there were no significant changes in phospholipid and inositol phosphate contents.     

Myelin P0 Glycoprotein: Identification of the Site Phosphorylated In Vitro and In Vivo by Endogenous Protein Kinases

Abstract: Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [³²P]PO₄³⁻ from [γ-³²P]ATP into protein constituents. Among these, P₀ glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P₀ protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two ³²P-phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P₀ sequence revealed, besides a Lys¹⁷² to Arg substitution, that in the first peptide, two serine residues (Ser¹⁷⁶ and Ser¹⁸¹) were phosphorylated, Ser¹⁷⁶ appearing to be modified subsequently to Ser¹⁸¹. In the second peptide, Ser¹⁹⁷, Ser¹⁹⁹, and Ser²⁰⁴ were phosphorylated. All these serines are clustered in the C-terminal region of P₀ protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P₀. We found that, in vivo, Ser¹⁸¹ and Ser¹⁷⁶ are not phosphorylated, whereas Ser¹⁹⁷, Ser¹⁹⁹, Ser²⁰⁴, Ser²⁰⁸, and Ser²¹⁴ are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level.     

Cysteine Sulfinic Acid in the Central Nervous System: Uptake and Release of Cysteine Sulfinic Acid by a Rat Brain Preparation

Abstract: Uptake and release of cysteine sulfinic acid by synaptosomal fractions (P₂) and slices of rat cerebral cortex were investigated. The P₂ fraction had a Na⁺-dependent high-affinity uptake system for cysteine sulfinic acid (K_m, 12μM), which was restricted to the synaptosomes. High-affinity uptake of cysteine sulfinic acid was competitively inhibited by glutamate, aspartate, and cysteic acid. None of the various centrally acting drugs tested specifically inhibited this transport system. Release of [¹⁴C]cysteine sulfinic acid from preloaded cortical slices or P₂ fractions was examined by a superfusion method, which avoided reuptake of released [¹⁴C]cysteine sulfinic acid. High K⁺ (56 m M ) and veratridine (10μM) stimulated the release of cysteine sulfinic acid from slices and the P₂ fraction in a partly Ca²⁺-dependent manner. Diazepam at concentrations of 10 and 100 μM markedly inhibited the stimulated release, but not the spontaneous release, by cortical slices. On the contrary, it had no effect on the stimulated release of cysteine sulfinic acid from the P₂ fraction.     

Formation of a Disulfide Bond in the Immunoglobulin Domain of the Myelin P0 Protein Is Essential for Its Adhesion

Abstract: It is widely accepted, although never demonstrated, that the formation of a disulfide bond in the majority of immunoglobulin (Ig)-like domains stabilizes their final conformation and thus is essential to their functioning as adhesion/recognition molecules. The myelin P₀ protein, which has been shown directly to behave as a homophilic adhesion molecule, contains a single Ig-like domain, stabilized by a putative Cys²¹-Cys⁹⁸ disulfide bond. To test if this bond is indeed necessary to the adhesive function of P₀, the nucleotides in the P₀ cDNA coding for Cys²¹ were altered to code for an alanine. The mutated P₀ cDNA was transfected into Chinese hamster ovary cells, expression of the mutated P₀ protein was characterized, and the adhesiveness of Cys²¹-mutated P₀-expressing cells and that of cells expressing equivalent surface amounts of the unmutated protein were compared. It was found, as we previously reported, that incubation of a single cell suspension of the unmutated P₀-expressing cells resulted in the rapid formation of large aggregates. In contrast, after a similar incubation the cells expressing the Cys²¹-mutated P₀ were still mostly single cells, a result indistinguishable from that observed with the control transfected cells. This suggests that the P₀ protein, when mutated at Cys²¹, does not behave as a homophilic adhesion molecule, which in turn implies that the formation of an Ig domain disulfide bond is essential to the functioning of this molecule.     

Phosphorylation and Fucosylation of Myelin Protein In Vitro by Sciatic Nerve from Developing Rats

Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P₀, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [³²P]orthophosphate or [³H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [³²P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2-day-old rats could incorporate radioactive fucose and [³²P]-phosphate into several proteins at the P₀ region of polyacrylamide gels. Specific radioactivity of [³H]fucose in P₀ protein was highest in preparations from 5-day-old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [³²P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.     

Concanavalin A Receptors Associated with Rat Brain Synaptic Junctions Are High Mannose-Type Oligosaccharides

Abstract: Glycoproteins were isolated from a rat brain synaptic junction fraction by affinity chromatography on Concanavalin A-agarose. The isolated glycoproteins were digested with pronase and radiolabeled with ¹²⁵I-Bolton Hunter reagent, and ¹²⁵I-Concanavalin A-binding glycopeptides were isolated by chromatography on Concanavalin A-agarose. Treatment of the ¹²⁵I-Concanavalin A-binding glycopeptides with either α-mannosidase or endo-β- N -acetylglucosaminidase-C₁₁ abolished their interaction with Concanavalin A. The pronase digest was reacted with endo-β-N-acetylglucosaminidase-C₁₁ and released oligosaccharides were reduced with NaB³H₄. Following affinity chromatography on Concanavalin A-agarose, Concanavalin A-binding [³H]oligosaccharides were chromatographed on Biogel P₄. Two major oligosaccharides corresponding to standard carbohydrates containing eight and five mannose residues were identified. Treatment of these oligosaccharides with α-mannosidase converted them to smaller saccharides having a mobility on Biogel P₄ columns equal to the standard disaccharide mannose-β-1-4- N '-acetylglucosamine. These results demonstrate that the Concanavalin A receptor activity associated with CNS synaptic junctions resides in asparaginelinked oligosaccharides of the high-mannose type.     

The effect of variation of thermal processing on the microbial spoilage of chub-packed luncheon meat

Process pasteurization values for reference temperature 70°C (P₇₀) were calculated from the temperature profiles of 250 g luncheon meat chubs cooked under experimental conditions. A simple equation relating Process P₇₀-value and the time and temperature of cooking was derived. With minimal cooking (P₇₀= 40) the surviving microflora (10³/g) was dominated by species of Lactobacillus, Brochothrix and Micrococcus. These organisms were destroyed by more intensive cooking (P₇₀= 105), leaving a flora (10²/g) composed of Bacillus and Micrococcus species. The spoilage that developed after 14 d storage at 25°C reflected the severity of the heat treatment received by each chub: with P₇₀ between 40 and 90, a Streptococcus spoilage sequence occurred; with P₇₀ between 105 and 120, a Bacillus/Streptococcus spoilage sequence occurred; with P₇₀ of 135 and above, a Bacillus spoilage sequence occurred. Cooking to a P₇₀= 75 was adequate to reduce the surviving microflora to the 10²/g level associated with current good manufacturing practice.