Stimulation of a cell-mediated cytotoxic response to FeLV-induced T cell lymphomas in the cat

Nonspecific cell-mediated cytotoxicity was examined in the peripheral blood and spleens of normal and vaccinia virus-infected adult domestic cats. Natural cytotoxic (NC)-like cells, as measured by lysis of vaccinia- or HSV-infected, adherent cat tongue cells, were found in both the spleen and peripheral blood of normal, nonimmune cats. Cytotoxicity was expressed in a 16-hr assay but not in a 4-hr assay. Natural killer (NK)-like cells, as measured by lysis of an FeLV-induced lymphoid tumor cell line (FL-74) growing in suspension, were found in the spleen but not PBL, and required a 16-hr assay for expression. Infection with vaccinia virus did not increase the activity of feline NC-like cells in either the peripheral blood or the spleen. NK-like function, however, was increased. Cytotoxicity peaked 6 days post-infection and required a 16-hr assay for maximal expression of cell lysis. Furthermore, a cell with cytotoxic characteristics of the spleen NK-like cell appeared at low levels in the circulation at 6 days post-vaccinia infection. NK-like cells from vaccinia-infected cats showed some cytotoxicity for FL74 targets in a 4-hr assay. The cat thus possesses at least two functionally different populations of naturally cytotoxic cells. NC-like cells are found in the spleen and peripheral blood, lyse virus-infected monolayer targets, and are not activated by infection. NK-like cells are found in the spleen, lyse-lymphoid tumor targets, and can be activated by infection, with their peak activity occurring 6 days after infection.     

Four-Hour Urease Test for Distinguishing Between Klebsiella and Enterobacter

Infections with Klebsiella and Enterobacter have increased among hospitalized patients. To study such infections, relatively simple but precise methods are needed for clinical laboratories to identify the two genera accurately. Moreover, a rapid identification is essential for assisting with the therapy of the patients. For this purpose, a new 4-hr urease test was developed so that colonies could be tested directly from blood-agar plates which have been inoculated with clinical material and allowed to incubate overnight. This 4-hr test was positive with 98.5% of 202 Klebsiella species and negative with 80 Enterobacter species. As a single criterion for distinguishing between the two major genera, the new 4-hr urease test was just as accurate as a motility test (99% of the 282 isolates were accurately identified with either). The 4-hr urease test represents a simple, rapid, and reliable technique which is ideally suited for use in clinical microbiology laboratories.     

Suppression of Fas-mediated signaling pathway is involved in zinc inhibition of ethanol-induced liver apoptosis

Apoptosis is critically involved in hepatic pathogenesis induced by acute alcohol exposure. This study was undertaken to test the hypothesis that zinc interferes with an important Fas ligand-mediated pathway in the liver, leading to the inhibition of ethanol-induced apoptosis. Male 129/Sv(PC)J mice were injected subcutaneously with ZnSO4 (5 mg of Zn ion/kg) in 12-hr intervals for 24 hr before intragastric administration of ethanol (5 g/kg) in 12-hr intervals for 36 hr. Ethanol-induced apoptosis in the liver was detected by a terminal deoxynucleotidyl transferase nick-end labeling assay and was further confirmed by electron microscopy. The number of apoptotic cells in the livers pretreated with zinc was significantly decreased, being only 15% of that found in the animals treated with ethanol only. Characteristic apoptotic morphological changes observed by electron microscopy were also inhibited by zinc. Importantly, zinc inhibited ethanol-induced activation of caspase-3, the primary executioner protease responsible for alcohol-induced liver apoptosis, and caspase-8 as determined by enzymatic assay. Immunohistochemical analysis revealed that zinc inhibited ethanol-induced endogenous Fas ligand activation, which is a key component in signaling pathways leading to hepatic caspase-8 and subsequent caspase-3 activation and apoptosis. These results demonstrate that zinc is a potent inhibitor of acute ethanol-induced liver apoptosis, and this effect occurs primarily through zinc interference with Fas ligand pathway and the suppression of caspase-3.     

Role of cytokines in the monocyte-mediated augmentation of human natural killer cell activity

Previous work has shown that normal human monocytes can augment natural killer (NK) cell activity both when mixed with enriched null cells in the assay and when precultured with enriched null cells and removed prior to testing. The data presented here show that a 4-hr preculture period is superior to slightly longer periods (10-12 hr) for demonstrating the augmentation. The role of cytokines in the monocyte effect was then investigated using a variety of antibody and recombinant reagents. Both monoclonal and rabbit polyclonal antibodies to IL-1 and IL-2 inhibited the monocyte effect, whereas antibodies against IFN-alpha and IFN-gamma from both sources had no effect. Of these cytokines, only IL-1 could be demonstrated (using a sensitive IL-1-dependent-IL-2 synthesis assay) in the supernatants of 4-hr cultures of monocytes plus null cells or null cells only. The ability to detect IL-1 was specifically inhibited by rabbit antibody to human IL-1 at 1:20 and 1:200 dilutions, but only the greater concentration inhibited the monocyte effect on NK activity. In contrast, the detection of soluble IL-1 was not inhibited by including monoclonal anti-IL-1 (1:20 dilution) in the 4-hr culture, although the same reagent abrogated the monocyte effect under these conditions. Recombinant IL-1 (up to 100 units/ml) did not augment NK activity either when added to the assay or when precultured for 4 hr with enriched null cells, whereas either recombinant IL-2 or monocytes were effective under these conditions. These results provide the first evidence for a cellular, and potentially physiologic, basis for the regulation of NK activity by IL-1 and IL-2, which had been previously known to act at pharmacologic levels in vitro.     

Induction of tumor cytotoxic immune cells using a protein from the bitter melon (Momordica charantia)

The fruit and seeds of the bitter melon (Momordica charantia) have been reported to have anti-leukemic and antiviral activities. This anti-leukemic and antiviral action was associated with an activation of murine lymphocytes. A partially purified protein factor from the bitter melon caused an infiltration and activation of peritoneal exudate cells in C57B1/6J, C3H/HeJ, and C3H/HeN mice. When the extract was injected twice a week at 8 micrograms of protein per ip injection for 0-4 weeks, the peritoneal exudate cells from the treated mice were cytotoxic in a long-term (18-hr) 51Cr-release assay against a range of labeled targets: L1210, P388, and MOLT-4 tumor cells. Cytotoxicity was also observed against YAC-1 targets in a short-term (4-hr) assay. Fractionation of the cytotoxic immune cells implicated a nonadherent cell population which was capable of killing an NK-sensitive cell line in a 4-hr 51Cr-release assay. Unit gravity sedimentation studies indicated that the cytotoxicity was due to either a neutrophil or a large lymphocyte. Antibody depletion experiments using antibody to asialo GM1, an NK cell-specific antibody, depleted cytotoxicity observed in nonadherent, Ficoll/Hypaque-separated PEC. This suggests that at least part of the anti-leukemic activity of the bitter melon extract is due to the activation of NK cells in the host mouse.     

Separation of rat leukocytes by countercurrent distribution in aqueous two-phase systems: II. Subpopulations which mediate selective and nonselective lysis of normal and colon carcinoma target cells in vitro

Spleen cells from WF rats immunized to allogeneic lymphoid cells or to syngeneic colon carcinomas and from unimmunized controls were separated by countercurrent distribution in aqueous two-phase systems. The cells were assayed for cytotoxicity to allogeneic fibroblasts or syngeneic colon carcinoma cells and to syngeneic fibroblasts in a 24-hr ⁵¹Cr-release assay. Cells from immunized rats which selectively lysed the specific target cells were repeatedly found in one area of the distribution separate from the majority of cells, which nonselectively lysed syngeneic fibroblasts as well. A similar subpopulation which nonselectively lysed all target cells assayed was recovered from the spleens of unimmunized rats. The cells were also assayed for the ability to lyse antibody-coated thymocytes in a 4-hr ⁵¹Cr-release assay. The peak of K cells was found to overlap partially that of cells with nonselective cytotoxicity.     

Prototype of a Fully Automated Device for Determination of Bacterial Antibiotic Susceptibility in the Clinical Laboratory

A completely automated system for the performance of antibiotic susceptibility tests in the clinical laboratory is described. With a modicum of personnel involvement, data on 40 specimens tested against 13 antibiotics are obtained every hour after an initial 3-hr period. The step by step explanation of the functioning of this prototype system, based on a thoroughly tested manual model, is presented. The system compares well with the standard diffusion test and has a potential for application to other endeavors of the clinical microbiology laboratory with a comparable saving in time and labor.     

The "lazy" NK cells of Chediak-Higashi syndrome

Natural killer (NK) function, measured in a short-term (4-hr) 51Cr-release assay, is profoundly depressed in circulating PBL of donors with Chediak-Higashi syndrome (CHS). In this study, we demonstrate that CHS NK cells can express relatively normal lytic function after prolonged exposure in vitro to high levels of activating as well as cytotoxic stimuli. After activation with the human cloned interferon (B1) for 24 hr, CHS NK cells have lytic activity comparable to unactivated normals in a 4-hr 51Cr-release assay. In addition, after 5 days of activation with mitomycin C-treated B cell lines, CHS NK cells have levels of activity similar to those of activated normals but are defective in generating cytotoxic cells capable of lysing the stimulator B cell. Even though CHS NK cells are defective in a 4-hr 51Cr-release assay, after 16 hr they enhance their killing capability 200 to 400-fold. In fact, after 16 hr of interaction with K562 target cells, CHS NK cells are capable of releasing NK soluble cytotoxic factors. These results are consistent with the hypothesis that CHS NK cells have all the necessary cellular structures and molecules required for them to function as lytic effector cells, but their lack of cytotoxic function is due to a relative refractoriness in initiating the post-binding lytic mechanism.     

Circadian rhythm of serotonin binding in rat brain--II. Influence of sleep deprivation and imipramine

Sleep deprivation (SD) modified the circadian rhythm of specific high affinity serotonin (5-HT) binding to rat brain membranes. In control rats a 24-hr rhythm was evident with a trough at 1000-1200 and a nadir at 0000. During the last 26 hr of a 49 hr SD period, trough and peak values were delayed by 4-6 hr. The 24-hr mean binding was significantly (P less than 0.001) different from that of controls. If sleep deprivation was followed by recovery sleep (RS), the normal rhythm of 5-HT binding was obtained already within 1 hr after SD. The effects of SD and RS were ascertained by plasma ACTH and corticosterone assay. No significant change in the hormone rhythms were observed through the mean plasma level of ACTH and corticosterone were enhanced to about 180 and 150%, respectively. Chronic treatment with the antidepressant imipramine resulted in a decrease of the 24-hr mean 5-HT binding by about 50% and a 2-hr delay of peak and trough values. Imipramine treatment decreased the peak value of 5-HT concentration at 1000 to about 65% and appears to abolish the rhythm of 5-HT concentration.     

CAS agar diffusion assay for the measurement of siderophores in biological fluids

We developed a simple and universal method, by modifying the universal CAS (Chrome azurol S) assay, measuring siderophores in various biological fluids. We named the assay as CAS agar diffusion (CASAD) assay. CAS plate devoid of nutrients was prepared by using Bacto-agar (1.5%, w/v) as a matrix. Holes with 5-mm-diameter were punched on the CAS agar plate. Each hole was added by 35 microl of the test fluids containing Desferal that was twofold serially diluted. After incubating at 37 degrees C or room temperature for 4-8 h, the size of orange haloes formed around the holes was measured. The size of orange haloes correlated well with the concentration of Desferal in all the biological fluids tested in this study. CASAD assay showed consistent results in wide pH range from 5 to 9. Addition of iron to the test fluids containing Desferal decreased the size of orange haloes in a dose-dependent manner, which suggests that the CASAD assay detects only iron non-bound siderophore. These results suggest that CASAD assay would serve as a simple, stable, and highly reproducible test for screening and quantitative siderophore analysis in biological fluids.     

Reorganization and stabilization of acetylcholine receptor aggregates on rat myotubes

Aggregation of acetylcholine receptors (AChRs) is an important early feature of the postsynaptic development of the vertebrae neuromuscular junction. At later stages of differentiation, aggregates are remodeled and stabilized. Aggregation of AChRs can be induced on rat myotubes in culture within 4 hr by treatment with embryonic pig brain extract (EBX). In this study, further sequential changes in the distribution of AChRs were followed by video-intensified fluorescence microscopy. These studies have revealed that groups of AChR aggregates that have formed after 4 hr in EBX are reorganized during the exposure to EBX for 20 additional hr to form a smaller number of larger, oval-shaped aggregates. We have named these two types of aggregates "4-hr aggregates" and "24-hr aggregates". This reorganization occurs by the expansion and merging of individual aggregates within a group, and by the incorporation of newly inserted AChRs. The 24-hr aggregates are an average of 15 times greater in area than 4-hr aggregates, and contain regions with an apparent AChR site density (fluorescence intensity) that is more than twice that of 4-hr aggregates. Electron microscopy of mapped 24-hr aggregates revealed that folded plasma membrane is associated with these regions, probably accounting for the elevated fluorescence. The 24-hr aggregates are more stable than 4-hr aggregates, as determined by their significantly slower disassembly after removal of EBX, elevation of temperature (38 degrees C), reduction of extracellular calcium levels (0.1 mM), or the addition of sodium azide (7 mM). This was determined by following disassembly both statistically (using fixed cultures) and by direct observations of living myotubes. These findings were confirmed by measuring the sequential changes in relative AChR site density over time in individual living myotubes. Thus, 24-hr aggregates form by the reorganization of 4-hr aggregates; exhibit a more regular, compact shape; and are more stable than 4-hr aggregates. These changes in AChR organization and aggregate stability resemble the changes occurring after the initial formation of junctional AChR aggregates during embryonic development, demonstrating additional similarities between this model system and the developing neuromuscular junction.     

A comparison of lysis mediated by Lyt 2+ TNP-specific cytotoxic-T-lymphocyte (CTL) lines with that mediated by rapidly internalized lymphotoxin-containing supernatant fluids: evidence for a role of soluble mediators in CTL-mediated killing

A series of Lyt 2+, trinitrophenyl (TNP)-specific T-cell lines are shown to lyse 51Cr-labeled target cells in an antigen-specific, H-2-restricted fashion in a 4-hr assay. These cells also produce lymphotoxin, in addition to other factors, upon stimulation with TNP-haptenated syngeneic splenocytes. A technique for introducing macromolecules into the cytoplasm of fibroblasts by inducing the cells to pinocytose the molecule in hypertonic medium, and then lysing the newly formed pinocytic vesicles with a mild hypotonic shock was used to assess the role of soluble mediators in the cytotoxic T lymphocyte (CTL)-mediated lytic process. The technique itself has little effect on cell growth rate or viability. A minimum of 24 hr, and more frequently 48-72 hr is required for lymphotoxin to manifest it's lethal effect when it is merely included in the culture medium of growing fibroblasts. In contrast, supernatant fluids from the Lyt 2+ cells kill 51Cr-labeled fibroblasts in a dose-dependent fashion during a 4-hr assay when they are rapidly internalized via the osmotic procedure. The data serve as preliminary evidence of a role for soluble mediators such as lymphotoxin in T-cell-mediated lysis, and suggest that the cytotoxic-T-cell lethal hit may include a mechanism for rapidly internalizing a toxin into appropriate target cells.     

Monoclonal antibody against K562 cell line accelerates killing of the target cells by large granular lymphocytes

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG_2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG_2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.     

Sulphate uptake and metabolism in the chrysomonad, monochrysis lutheri.

The intracellular concentration of inorganic 35SO4 in Monochrysis lutheri cells exposed to 0.513 mM Na235SO4 for up to 6-hr remained constant at about 0.038 mM. The exchange rate of this 35SO4 with the external unlabelled sulphate was negligible compared to the rate of influx across the plasmalemma (0.032 mu moles/g cells/hr). The flux of free 35SO4 to organic 35S was 0.029 mu moles/g cells/hr. Assuming an internal electrical potential in the cells of -70 mV, this intracellular concentration of inorganic 35SO4 was well in excess of that obtainable by passive diffusion as calculated from the Nernst equation. These results indicate that sulphate is accumulated by an active mechanism rather than by facilitated diffusion. Sulphate uptake appears to occur via a carrier-mediated membrane transport system which conforms to Michaelis-Menten type saturation kinetics with a Km of 3.2 X 10(-5) M and Vmax of 7.9 X 10(-5) mu moles sulphate/hr/10(5) cells. Uptake was dependent on a source of energy since the metabolic inhibitor CCCP almost completely inhibited uptake under both light and dark conditions and DCMU caused a 50% decrease in uptake under light conditions. Under dark conditions, uptake remained at about 80% of that observed under light conditions and was little affected by DCMU, indicating that the energy for uptake could be supplied by either photosynthesis or respiration. A charge and size recognition site in the cell is implied by the finding that sulphate uptake was inhibited by chromate and selenate but not by tungstate, molybdate, nitrate or phosphate. Chromate did not inhibit photosynthesis. Cysteine and methionine added to the culture medium were apparently capable of exerting inhibition of sulphate uptake in both unstarved and sulphate-starved cells. Cycloheximide slightly inhibited sulphate uptake over an 8-hr period indicating, either a slow rate of entry of the inhibitor into the cells or a slow turnover of the protein(s) associated with sulphate transport.     

STAPHYOGRAM, a new rapid identification kit for the aerobic, gram-positive, catalase-positive cocci--application of fluorometric microplate hybridization for the pre-identification of 386 isolates used

A new simplified test kit, STAPHYOGRAM plate, was developed for 4-hr identification of aerobic, Gram-positive and catalase-positive cocci. The plate has 18 wells, in which different dehydrated substrates and nutrients are fixed. An 18-hr agar-culture suspension of a test strain with a turbidity of McFarland No. 4 was distributed into all wells in 50-microliters quantities. After 4-hr incubation at 37C, the profile number was obtained by summarizing positive reactions. The ability of the plate to differentiate the type strains of the 30 species of the three genera in the family Micrococcaceae was confirmed. These three genera are Staphylococcus, Micrococcus and Stomatococcus. The applicability of the fluorometric microplate hybridization technique to identification of aerobic, Gram-positive and catalase-positive cocci was confirmed by homologous hybridization among the type strains of the 30 species. Thus, 386 isolates of human and animal origin were pre-identified by microplate hybridization and used for evaluating the STAPHYOGRAM plate. Of the 236 profile numbers thus obtained with the 386 isolates, 218 (92.4%) were species-proper each and all for the 15 species of Staphylococcus and Stomatococcus mucilaginosus. A total of 342 (88.6%) of the 386 isolates were given such profile numbers, and were identified without any additional test. Among the 15 species identified primarily by the results of STAPHYOGRAM plate culture, S. caprae, S. lugdunensis, S. gallinarum and S. delphini were validly published after Approved Lists of Bacterial Names. The identified strains of S. caprae (48), S. haemolyticus (46), S. capitis (35) numbered between those of S. epidermidis (67) and S. saprophyticus (31). Profile numbers common to two species were seven (27 strains) and that to four species was one (17 strains). These 44 strains were identified with one to three additional tests. From these results, we were convinced that the STAPHYOGRAM test plate is useful for the rapid identification of members of family Micrococcaceae. By compiling STAPHYOGRAM plate data on genetically identified strains, an exclusive list of profile numbers will soon be prepared for perfection of the kit.     

Modified Microbiological Assay for Rapid Estimation of Antibiotic Concentrations in Human Sera

Antibiotic concentrations in human sera were estimated in 5 to 6 hr by a modified microbiological assay. By using Staphylococcus aureus and Streptococcus pyogenes as the assay organisms, the seeded assay plates were preincubated for 2 to 6 hr and then were stored at 4 C until used for assay. Paper discs saturated with the specimen were placed on the preincubated assay plates with reference discs saturated with known concentrations of antibiotic. After 5 to 6 hr of incubation, zones of antibacterial activity were measured and compared with a standard curve for estimation of antibiotic concentration. Results from this rapid assay method compared favorably with those from the commonly used 24-hr assay.     

Circadian rhythms of blood clotting time and coagulation factors II, VII, IX and X in rats

The 24-hr variations in clotting times and vitamin K-dependent blood coagulation factors were studied in rats kept on a 12-hr light-dark cycle (light on: 0600-1800 hours). Clotting times were determined under a binocular microscope by measuring the time required for the formation of the first fibrin thread. Factors II, VII and X were analyzed by the prothrombin test while the factor IX was quantified using the activated partial thromboplastin time assay. Results indicated that the clotting times were significantly longer during the dark (activity) period with a peak at 1:00 and a trough at 17:00. Similarly, a variation was found in factor activity levels: prothrombin (II), factor VII and factor X had higher activities during the light span (rest period). The highest activities found at 13:00 and 09:00 were statistically different from the minimum activity levels obtained at 21:00. Factor IX did not show a significant circadian variation.     

Density gradient fractionation of effector cells in human natural cell-mediated cytotoxicity

In order to separate and characterize cytotoxic effector cells in natural cell-mediated cytotoxicity (NCMC), human lymphocytes were fractionated by Percoll continuous density gradient centrifugation (Pharmacia, Piscataway, N.J.). Lymphocytes from normal donors were fractionated through a 35-ml gradient and 2- or 3-ml aliquots were collected, counted, and grouped into three or more fractions in order to obtain sufficient cells for testing. Fractions of cells were tested for cytotoxicity in a 4-hr chromium release test and/or a 40-hr [³H]proline assay. Cell markers were assessed by testing for cells forming E rosettes, EA rosettes, and for cells with surface membrane immunoglobulin (SMIg). The lightest fraction contained larger cells and also usually contained the highest concentrations of cells with receptors for the Fc portion of IgG (FcR + cells), although slight variations were seen among individual donors. Results of cytotoxicity tests showed that cells from the top portions of the Percoll gradient had consistently greater cytotoxic activity on a per cell basis than the denser cells sedimenting lower. Estimation of cytotoxic activity in lytic units showed that 54–75% of the activity was recovered in the top 26–29% of the cells. This approach to investigating cell-mediated cytotoxicity should yield useful information regarding cellular interaction in, and regulation of, cytotoxic activities.     

Evaluation of a New Incubation-Type Indole Strip Test

A total of 189 of Enterobacteriaceae and three strains of Clostridium perfringens were tested with a new incubation-type indole paper strip. This 4-hr incubation test gave the same results as a standard 48-hr indole test, except with some strains of Proteus rettgeri. Results with P. rettgeri varied somewhat according to the medium upon which the organisms had been grown.     

Metabolism of biotin and analogues of biotin by microorganisms. II. Further studies on the conversion of D-biotin to biotin vitamers by Lactobacillus plantarum

Birnbaum, Jerome (University of Cincinnati, Cinncinati, Ohio), and Herman C. Lichstein. Metabolism of biotin and analogues of biotin by microorganisms. II. Further studies on the conversion of d-biotin to biotin vitamers by Lactobacillus plantarum. J. Bacteriol. 92:913-919. 1966.-Lactobacillus plantarum growing in excess biotin converts a portion to two vitamers (combinable and uncombinable with avidin) not utilizable for growth. These were detected by differential yeast-lactobacillus assay. In the present study, suspensions of 12- and 72-hr cells showed no converting activity. Vitamer formation by nonproliferating 24-hr cells required glucose and exhibited a lag; 17-hr cells showed neither a lag nor a glucose requirement. Iodoacetate and chloramphenicol inhibited vitamer formation by 24-hr cells, but had no effect on 17-hr cells. Addition of hydrolyzed casein or preincubation in biotin decreased the lag and enhanced vitamer formation in 24-hr cells, but had no effect in 17-hr cells. Apparently, 17-hr cells contain the converting enzymes which degenerate as growth proceeds; the lag exhibited by 24-hr cells represents the time necessary to reform the enzymes. Equal amounts of the two vitamers were formed in 17-hr cells; only the avidin-combinable form was produced initially by 24-hr cells, unless hydrolyzed casein was present. Electrophoresis revealed that the avidin-combinable vitamer has the same charge as biotin,whereas the uncombinable form possesses both positive and negative groups. Column chromatography was used to separate the avidin uncombinable material from biotin and the avidin-combinable form. L. plantarum was unable to accumulate the avidin-uncombinable vitamer under conditions permitting good biotin accumulation. It was concluded that L. plantarum sequentially converts biotin to avidin-combinable and -uncombinable vitamers, the latter being impermeable to the cells.