Genome plasticity in the distal tail fiber locus of the T-even bacteriophage: recombination between conserved motifs swaps adhesin specificity

The adsorption specificity of the T-even phages is determined by the protein sequence near the tip of the long tail fibers. These adhesin sequences are highly variable in both their sequence and specificity for bacterial receptors. The tail fiber adhesin domains are located in different genes in closely related phages of the T-even type. In phage T4, the adhesin sequence is encoded by the C-terminal domain of the large tail fiber gene (gene 37), but in T2, the adhesin is a separate gene product (gene 38) that binds to the tip of T2 tail fibers. Analysis of phage T6 and Ac3 sequences reveals additional variant forms of this locus. The tail fiber host specificity determinants can be exchanged, although the different loci have only limited homology. Chimeric fibers can be created by crossovers either between small homologies within the structural part of the fiber gene or in conserved motifs of the adhesin domain. For example, the T2 adhesin determinants are flanked by G-rich DNA motifs and exchanges involving these sequences can replace the specificity determinants. These features of the distal tail fiber loci genetically link their different forms and can mediate acquisition of diverse host range determinants, including those that allow it to cross species boundaries and infect taxonomically distant hosts.     

Activity patterns in Glossina longipennis: a field study using different sampling methods

Protein folding in the cell is controlled at the levels of translation and post-translational modification, depends on a number of conserved proteins known as chaperones, and is catalyzed by specific enzymes, such as protein disulfide isomerase and peptidyl prolyl cis-trans isomerase. The chaperones stabilize folding intermediates and participate in assembly and disaggregation of supramolecular structures. Bacteriophage T4 is an especially convenient system for studying of protein folding mechanisms, since its genome encodes several virus-specific chaperones. In this review, the chaperones of phage T4 that take part in capsid formation (gp31 and gp40) and in folding and assembly of virion tail fibers (gp38, gp57A) have been considered. Protein encoded by gene 31 completely substitutes co-chaperonin GroES of the host cell in folding of the major capsid protein, gp23, aided by chaperonin GroEL. The product of gene 40, which is homologous to analogs of eukaryotic GroEL and peptidyl prolyl cis-trans isomerase, participates in assembly of gp20 while the formation of procapsid connector. The chaperone encoded by gene 57A is essential for folding and oligomerization of both long and short phage tail fibers. gp38, together with gp57A, participates in the formation of the distal part of the long fibers. This protein seems to represent a principally new group of chaperones that change steric structure of folded polypeptide. One phage chaperone, fibritin, encoded by gene wac (whiskers antigen control) and taking part in assembly the subunits of the long tail fibers is a constituent of the virion. Fibritin is a convenient model for studying mechanisms of folding and oligomerization of fibrous proteins due to its labile triple-stranded alpha-helical coiled-coil structure.     

Targeting of a short peptide derived from the cytoplasmic tail of the G1 membrane glycoprotein of Uukuniemi virus (Bunyaviridae) to the Golgi complex

Members of the Bunyaviridae family acquire an envelope by budding through the lipid bilayer of the Golgi complex. The budding compartment is thought to be determined by the accumulation of the two heterodimeric membrane glycoproteins G1 and G2 in the Golgi. We recently mapped the retention signal for Golgi localization in one Bunyaviridae member (Uukuniemi virus) to the cytoplasmic tail of G1. We now show that a myc-tagged 81-residue G1 tail peptide expressed in BHK21 cells is efficiently targeted to the Golgi complex and retained there during a 3-h chase. Green-fluorescence protein tagged at either end with this peptide or with a C-terminally truncated 60-residue G1 tail peptide was also efficiently targeted to the Golgi. The 81-residue peptide colocalized with mannosidase II (a medial Golgi marker) and partially with p58 (an intermediate compartment marker) and TGN38 (a trans-Golgi marker). In addition, the 81-residue tail peptide induced the formation of brefeldin A-resistant vacuoles that did not costain with markers for other membrane compartments. Removal of the first 10 N-terminal residues had no effect on the Golgi localization but abolished the vacuolar staining. The shortest peptide still able to become targeted to the Golgi encompassed residues 10 to 40. Subcellular fractionation showed that the 81-residue tail peptide was associated with microsomal membranes. Removal of the two palmitylation sites from the tail peptide did not affect Golgi localization and had only a minor effect on the association with microsomal membranes. Taken together, the results provide strong evidence that Golgi retention of the heterodimeric G1-G2 spike protein complex of Uukuniemi virus is mediated by a short region in the cytoplasmic tail of the G1 glycoprotein.     

A biotechnological method provides access to aggregation competent monomeric Alzheimer's 1-42 residue amyloid peptide

Senile plaques, a neuropathological hallmark of Alzheimer's disease, consist primarily of insoluble aggregates of beta-amyloid peptide (A beta). A 42-residue peptide (A beta 1-42) appears to be the predominant form. In contrast to A beta 1-40, A beta 1-42 is characterized by its extreme tendency to aggregate into fibers or precipitate. A tailored biotechnological method prevents aggregation of A beta 1-42 monomers during its production. The method is based on a protein tail fused to the amino terminus of A beta. This tail leads to a high expression in E. coli, and a histidine affinity tag facilitates purification. Selective cleavage of the fusion tail is performed with cyanogen bromide by immobilizing the fusion protein on a reversed phase chromatography column. Cleavage then occurs only at the methionine positioned at the designed site but not at the methionine contained in the membrane anchor sequence of A beta. Furthermore, immobilization prevents aggregation of cleaved A beta. Elution from the HPLC column and all succeeding purification steps are optimized to preserve A beta 1-42 as a monomer. Solutions of monomeric A beta 1-42 spontaneously aggregate into fibers within hours. This permits the investigation of the transition of monomers into fibers and the correlation of physico-chemical properties with biological activities. Mutations of A beta 1-42 at position 35 influence the aggregation properties. Wild-type A beta 1-42 with methionine at position 35 has similar properties as A beta with a methionine sulfoxide residue. The fiber formation tendency, however, is reduced when position 35 is occupied by a glutamine, serine, leucine, or a glutamic acid residue.     

Phosphatidylinositol 3-kinase is necessary and sufficient for insulin-stimulated stress fiber breakdown

Rat-1 fibroblasts overexpressing the human insulin receptor undergo rapid actin rearrangement in response to insulin. Breakdown of stress fibers present in quiescent cells is followed by transient membrane ruffling and a return of stress fibers. We investigated the signaling pathways that mediate this insulin-stimulated reorganization of the actin cytoskeleton, which was visualized with rhodamine-phalloidin. Treatment of cells with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin prevented insulin action at the preliminary step of stress fiber breakdown. Cellular microinjection of a polyclonal antibody directed against the p85 subunit of PI3-kinase as well as a purified recombinant p85-SH2 domain protein also inhibited actin reorganization. Transient expression of a constitutively active form of PI3-kinase (p110*) was sufficient to cause both stress fiber breakdown and membrane ruffling in the absence of insulin. Microinjection of a polyclonal anti-Shc antibody or dominant negative N17-Ras protein did not affect actin dynamics, and although constitutively active V12-Ras caused modest cytoskeletal reorganization, this effect was blocked by pretreatment with wortmannin. In summary, activation of PI3-kinase is necessary and sufficient to stimulate actin rearrangement, indicating that PI3-kinase may initiate the only signaling cascade required for insulin to induce cytoskeletal restructuring.     

Sak57, an acidic keratin initially present in the spermatid manchette before becoming a component of paraaxonemal structures of the developing tail

We have previously reported that Sak57 (for Spermatogenic cell/Sperm-associated keratin of molecular mass 57 kDa) is an acidic keratin found in rat spermatocytes, spermatids, and sperm. Sak57 displays conserved amino acid sequences found in the 1A and 2A regions of the alpha-helical rod domain of keratins in human, rat, and mouse. We now report indirect immunofluorescence, confocal laser scanning microscopy and immunogold electron microscopy data showing that Sak57 is associated with the microtubular mantie of the manchette, a transient microtubular structure largely regarded as formed by tubulin and microtubule-associated proteins. The immunocytochemical localization of Sak57 was detected with a polyclonal antiserum to a multiple antigenic peptide (MAP) containing an amino acid sequence known to be present in the 2A region of the alpha-helical rod domain. During spermiogenic steps 8-12, Sak57 immunoreactive sites were restricted to microtubular mantie of the manchette which encircles the spermatid nucleus during shaping and chromatin condensation. At later stages (spermiogenic steps 12-14), Sak57 immunoreactive sites in the spermatid head region disappeared gradually as specific immunoreactivity appeared along the already assembled axoneme of the developing spermatid tail. Immunogold electron microscopy confirmed the presence of Sak57 immunoreactivity among microtubules of the manchette and on outer dense fibers and the longitudinal columns linking the ribs of the fibrous sheath. Mature spermatids (spermiogenic step 19) displayed tails with an immunofluorescent banding pattern contrasting with the lack of Sak57 immunoreactivity in the head region. Results from this study suggest that, during early spermiogenesis, a microtubular-Sak57 scaffolding is associated with the spermatid nucleus during shaping and chromatin condensation. During late spermiogenesis, the dispersion of the manchette coincides with the progressive visualization of Sak57 in the paraaxonemal outer dense fibers and longitudinal columns of the fibrous sheath in the developing spermatid tail.     

Mutation of protease domain residues Lys37-39 in human protein C inhibits activation by the thrombomodulin-thrombin complex without affecting activation by free thrombin

Activated protein C (aPC) is an important feedback regulator of the clotting cascade. In vivo, the conversion of protein C (PC) from its zymogen to activated form is mediated primarily by thrombin bound to thrombomodulin (TM), an endothelial cell surface protein. Molecular modeling suggests residues Lys37-Lys38-Lys39 of protein C's serine protease domain reside in a surface-exposed loop (variable region 1) whose high concentration of positive charge might be involved in protein-protein interactions. In this study, we have examined the role of the conserved tribasic Lys37-39 charge center in human protein C activation. This sequence was changed to acidic by substitution with Asp37-Glu38-Asp39 (DED) and Glu37-Glu38-Glu39 (EEE), or to neutrality by substitution with Gly37-Gly38-Gly39 (GGG). These mutant PCs, expressed and purified from recombinant human 293 cells, appeared normal with regard to intracellular processing, ability to be secreted, and formation of a viable active site for tripeptidyl-p-nitroanilide substrate cleavage. For activation by free thrombin, wild-type (wt) and mutant PCs displayed equivalent activation rates, as well as identical calcium-dependent inhibition of such activation. Activation of wt-PC with a soluble TM-thrombin complex yielded a 2,000-fold faster rate compared with that by free thrombin at the same (physiological) calcium level. In contrast, the acidic mutants DED and EEE exhibited virtually no TM-mediated increase in activation rate, while the neutral mutant GGG was somewhat intermediate with a 30-fold stimulation of activation rate. These reductions in activation rate were independent of the presence of chondroitin sulfate on TM. Our observations represent the first identification of residues whose mutation essentially uncouples activation by the TM-thrombin complex without affecting activation by free thrombin. Further, our results suggest that VR1 residues within the zymogen form of a serine protease can be important for recognition by physiological activators.     

The narcotic properties of xenon and the prospects for its use in anesthesiology

An alkaline phosphatase was purified from conidia of a Neurospora crassa wild type strain. The M(r) of the purified native enzyme was estimated as ca 145,000 and 110,000 by gel filtration, in the presence and absence of magnesium ions, respectively. A single polypeptide band of M(r) 36,000 was detected by SDS-PAGE, suggesting that the native enzyme was a tetramer of apparently identical subunits. Conidial alkaline phosphatase was an acidic protein (pl = 4.0 +/- 0.1), with 40% carbohydrate content. Optimal pH was affected by substrate concentration and magnesium ions. Low concentrations of calcium ions (0.1 mM) had slight stimulatory effects, but in excess (5 mM) caused protein aggregates with decreased activity. The enzyme specificity against different substrates was compared with those reported for constitutive or Pi-repressible alkaline phosphatases produced by N. crassa. The results suggested that the conidial alkaline phosphatase represented a different class among other such enzymes synthesized by this organism.     

Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics

Analysis of antimicrobial activities that are present in the skin secretions of the South American frog Phyllomedusa bicolor revealed six polycationic (lysine-rich) and amphipathic alpha-helical peptides, 24-33 residues long, termed dermaseptins B1 to B6, respectively. Prepro-dermaseptins B all contain an almost identical signal peptide, which is followed by a conserved acidic propiece, a processing signal Lys-Arg, and a dermaseptin progenitor sequence. The 22-residue signal peptide plus the first 3 residues of the acidic propiece are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The 25-residue amino-terminal region of prepro-dermaseptins B shares 50% identity with the corresponding region of precursors for D-amino acid containing opioid peptides or for antimicrobial peptides originating from the skin of distantly related frog species. The remarkable similarity found between prepro-proteins that encode end products with strikingly different sequences, conformations, biological activities and modes of action suggests that the corresponding genes have evolved through dissemination of a conserved "secretory cassette" exon.     

Purification, and biochemical and biological characterization of an immunosuppressive and lymphocyte mitogenic protein secreted by Streptococcus sobrinus

An immunosuppressive/mitogenic (ISM) protein was purified from the supernatants of cultures of Streptococcus sobrinus with an isoelectric point of 4.75 and a relative molecular mass of 38 kDa (p38). Treatment of C57BL/6 mice with p38 induced an increase in the numbers of non-specific splenic Ig-secreting plaque-forming cells (PFC) with peak responses on day 3 for IgM-secreting PFC and on day 5 for IgG-secreting PFC, with an isotype pattern consisting predominantly of IgG2a and IgG2b. This increase was accompanied by a lymphocyte blastogenic response of both T and B lymphocytes. The in vitro effects of p38 on pure B, T and total splenic lymphocytes indicated that this ISM protein was primarily a B cell mitogen, being T cells activated subsequently by the generation of B blasts. Suppression of the murine primary immune response against sheep red blood cells was observed in C57BL/6 mice treated 4 days before with p38. The amino acid sequence of the N-terminus of p38 has a significant similarity with several enolases, particularly with rabbit enolase. However, the biological effects ascribed to p38 have not been detected after in vivo treatment with that enolase. The immunosuppressive effect of p38 was abrogated by depletion of IL-10 but not of IL-4. In agreement with this observation IL-10 was the only cytokine detected in serum of C57BL/6 mice after p38 treatment and the peak of serum levels was observed as soon as 2 h after treatment.     

Decreased proportion of type I myofibers in skeletal muscle of dogs with chronic heart failure

BACKGROUND: Whether biochemical and histological abnormalities of skeletal muscle (SM) develop in patients with chronic heart failure (HF) remains controversial. In the present study, dogs with chronic HF were used to examine potential alterations of SM fiber type, fiber size, number of capillaries per fiber (C/F), beta-adrenergic receptor density (Bmax), and fiber ultrastructural integrity. METHODS AND RESULTS: HF was produced in 17 dogs by sequential intracoronary microembolizations. Biopsies of the lateral head of the triceps muscle were used in all studies. Type I and type II fibers were differentiated by myofibrillar ATPase (pH 9.4 or 4.2). Bmax was assessed by radioligand binding and SM ultrastructure by transmission electron microscopy. Comparisons were made with biopsies obtained from nine control dogs. The percentage of SM type I fibers was reduced in HF dogs compared with control dogs (19 +/- 2% versus 32 +/- 5%) (p < 0.001), whereas the percentage of SM type II fibers was increased (81 +/- 2% versus 68 +/- 5%) (p < 0.001). The change in fiber type composition was not associated with a preferential atrophy or hypertrophy of either fiber type. There was no difference in SM Bmax (198.9 +/- 14.3 versus 186.8 +/- 17.3 fmol/mg protein) or in C/F (5.37 +/- 0.26 versus 5.84 +/- 0.21) between HF dogs and control dogs. No ultrastructural abnormalities were present in SM fibers of HF dogs. CONCLUSIONS: In dogs with HF, there is a decrease in the relative composition of the slow-twitch type I SM fibers and an increase in fast-twitch type II fibers. The shift in fiber type composition is not associated with preferential atrophy of either fiber type or with a reduction in C/F, beta-adrenergic receptor density, or structural abnormalities of the myofibers.     

ATP hydrolysis catalyzed by human replication factor C requires participation of multiple subunits

Human replication factor C (hRFC) is a five-subunit protein complex (p140, p40, p38, p37, and p36) that acts to catalytically load proliferating cell nuclear antigen onto DNA, where it recruits DNA polymerase delta or epsilon to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomplex of hRFC consisting of three subunits (p40, p37, and p36) contained DNA-dependent ATPase activity. However, it is not clear which subunit(s) hydrolyzes ATP, as all five subunits include potential ATP binding sites. In this report, we introduced point mutations in the putative ATP-binding sequences of each hRFC subunit and examined the properties of the resulting mutant hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduced replication activity of the hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in the ATP binding site of the p38 subunit did not alter the replication activity of hRFC. These findings indicate that the replication activity of hRFC is dependent on efficient ATP hydrolysis contributed to by the action of four hRFC subunits.     

Surveillance of Pseudomonas aeruginosa in intensive care units: clusters of nosocomial cross-infection and encounter of a multiple-antibiotic resistant strain

Fiber length has been implicated as a determinant of fiber toxicity. Fibers of narrowly defined length can be generated by dielectrophoretic classifiers. Since the quantities of fibers produced are very small, we developed a rat alveolar macrophage microculture system to study the toxicity of these samples. The objective of this study was to examine the role of fiber length on the cytotoxicity of Manville code 100 (JM-100) fibers. Rat alveolar macrophages were cultured with 0-500 microg/ml of 5 lengths of JM-100 fibers on 96-well plates. After 18 h, well supernatants were removed and lactate dehydrogenase (LDH) activity was measured to assess cell damage. Chemiluminescence (CL), an assessment of macrophage function, was measured by adding lucigenin with or without zymosan, a particulate stimulus, to appropriate wells. For each fiber length the effects were concentration dependent: CL declined and LDH rose with increasing fiber concentration. Comparing the effects of different lengths showed the greatest toxicity from a relatively long fiber sample (mean length = 17 microm). Microscopic examination of the interaction of fibers with macrophages revealed multiple macrophages attached along the length of the long fibers. This suggests that frustrated, or incomplete, phagocytosis may be a factor in the increased toxicity of longer fibers. Overall the results demonstrate that length is an important determinant of toxicity for JM-100 fibers.     

Translocation of N-terminal tails across the plasma membrane

Previously we have shown that the first hydrophobic domain of leader peptidase (lep) can function to translocate a short N-terminal 18 residue antigenic peptide from the phage Pf3 coat protein across the plasma membrane of Escherichia coli. We have now examined the mechanism of insertion of N-terminal periplasmic tails and have defined the features needed to translocate these regions. We find that short tails of up to 38 residues are efficiently translocated in a SecA- and SecY-independent manner while longer tails are very poorly inserted. Efficient translocation of a 138 residue tail is restored and is Sec-dependent by the addition of a leader sequence to the N-terminus of the protein. We also find that while there is no amphiphilic helix requirement for N-terminal translocation, there is a charge requirement that is needed within the tail; an arginine and lysine residue can inhibit or completely block translocation when introduced into the tail region. Intriguingly, the membrane potential is required for insertion of a 38 residue tail but not for a 23 residue tail.     

Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins

The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family despite the fact that they encode end products having very different biological activities. These genes might contain a homologous export exon comprising the 5'-untranslated region, the 22-residue signal peptide, the 20-24-residue acidic spacer, and the basic pair Lys-Arg.     

Muscle pathology and clinical measures of disability in children with cerebral palsy

We performed a histologic and morphometric study of spastic muscle from 10 children with diplegic cerebral palsy, comparing muscle structure with the gait parameters of energy expenditure index and dynamic electromyography. Variations in fiber area within and between fiber types were increased significantly in children with cerebral palsy. In each of the control subjects, the combined coefficient of variation for type-1 and type-2 fiber area was less than 25% and the average was 17%; in the subjects with cerebral palsy, the combined coefficient of variation was more than 25% and the average was 36% (p < or = 0.004). The average difference between the mean area of type-1 and type-2 fibers was 26.7 +/- 18.9% for subjects with cerebral palsy and 4.2 +/- 2.4% for control subjects (p < or = 0.004). There was a 67% predominance of one fiber type in the subjects with cerebral palsy compared with a 55% predominance in the control subjects (p < or = 0.03). The difference between the total area of type-1 and type-2 fibers was 57% in the subjects with cerebral palsy and 17% in the control subjects (p < or = 0.002). There was a significant correlation between the combined coefficient of variation of fiber area and the energy expenditure index (r = 0.77, p < or = 0.03). The difference between the mean area of type-1 and type-2 fibers correlated with prolongation of electromyographic activity (r = 0.69, p < or = 0.05). No abnormalities in fiber ultrastructure were found in the subjects with cerebral palsy. Children with cerebral palsy had abnormal variation in the size of muscle fibers and altered distribution of fiber types. The values for variation in fiber area correlated with the energy expenditure index and with prolongation of electromyographic activity during walking.     

Encapsulation of OKT3 cells in hollow fibers

Encapsulation of an OKT3 cell line in hollow fibers was evaluated in vitro and in vivo. The cell line is a mouse hybridoma producing immunoglobulin G2a (IgG2a) against CD3 human T lymphocytes and thus may function as a nonspecific activation system of a subpopulation of human T lymphocytes. For encapsulation purpose, hollow fibers of polypropylene K600 PP Accurel (Akzo, Germany) were selected. Hollow fibers were siliconized to improve membrane biocompatibility for in vivo experiments. The siliconized hollow fibers exhibited acceptable diffusive permeability (P) [ml/min/m2] for small solutes (for creatinine, p = 63.9 +/- 2.0, n = 3) and larger solutes (for albumin, p = 16.9 +/- 1.9, n = 3; for IgG, p = 1.0 +/- 0.2, n = 3). The 12 cm long hollow fibers were filled with a suspension of OKT3 cells of an average density of 10(6) cells/ ml, and both ends were sealed. The encapsulated cells were cultivated in RPMI 1640/10% CS medium at 37 degrees C, 5% CO2 for a period of 3 to 4 days. After the culture period, the medium was tested on human peripheral blood lymphocytes for the presence of anti-CD3 antibody and read in a flow FACS-trac cytometer (Becton Dickinson Immunocytochemistry Systems, San Diego, CA). The tightness of hollow fiber sealing was tested with a bubble point method. The number of cells increased after cultivation by four- to nine-fold on average (n = 11). Ten experiments were performed in vivo with OKT3 cells encapsulated in hollow fibers and implanted subcutaneously into mice for 3 days. In 50% of the experiments, some anti-CD3 antigens on human lymphocytes were found; however, the difference, in comparison with control, in percent of CD3+ was insignificant. In conclusion, the hollow fiber method for cultivation of hybridoma cells in vitro allows for separation of cells from the medium containing secreted anti-CD3 antibodies and is effective in maintaining cell viability. In vivo application needs additional study.