缺氧复氧诱导人肾小管上皮细胞蛋白质组表达变化的初步研究

目的:采用将人肾小管上皮细胞置于缺氧环境中然后复氧的方法模拟缺血再灌注(ischemia reper-fusion,IR)所致肾损伤的发生和发展过程,研究IR对人肾小管上皮细胞表达蛋白质组的影响。方法:人肾小管上皮细胞株(HK-2细胞)常规培养,随机分为2组,IR组缺氧培养8h,然后复氧培养24h,对照组常规培养,裂解细胞,提取全细胞蛋白。双向电泳(2-DE)分离,ImageMaster2D Platinum V5.0软件进行差异表达蛋白质组分析,基质辅助激光解吸附离子化飞行时间质谱(MALDI-TOF-MS)鉴定蛋白质。结果:通过对2-DE图谱蛋白斑点的匹配及对比分析,与HK-2缺氧复氧诱导的差异表达蛋白斑点为109个;经质谱鉴定的7个差异表达的蛋白斑点包括:核糖体蛋白S2、普通转录因子ⅡH亚基1变体、双链断裂修复蛋白rad-21同源物、富含亮氨酸重复序列蛋白-45、DNA依赖性蛋白激酶、丝/苏氨酸蛋白激酶Chk1和程序性细胞死亡蛋白-4。结论:IR组与对照组的表达蛋白质组相比较存在明显差异;7个与HK-2缺氧复氧诱导表达改变的蛋白质的功能涉及细胞增殖、细胞凋亡、细胞信号转导、抗细胞损伤等,提示IR的发生发展与相应的病理生理过程相关。     

Investigating the Role of Ischemia vs. Elevated Hydrostatic Pressure Associated with Acute Obstructive Uropathy

Obstructive uropathy can cause irreversible renal damage. It has been hypothesized that elevated hydrostatic pressure within renal tubules and/or renal ischemia contributes to cellular injury following obstruction. However, these assaults are essentially impossible to isolate in vivo. Therefore, we developed a novel pressure system to evaluate the isolated and coordinated effects of elevated hydrostatic pressure and ischemic insults on renal cells in vitro. Cells were subjected to: (1) elevated hydrostatic pressure (80 cm H₂O); (2) ischemic insults (hypoxia (0% O₂), hypercapnia (20% CO₂), and 0 mM glucose media); and (3) elevated pressure + ischemic insults. Cellular responses including cell density, lactate dehydrogenase (LDH) release, and intracellular LDH (LDH_i), were recorded after 24 h of insult and following recovery. Data were analyzed to assess the primary effects of ischemic insults and elevated pressure. Unlike pressure, ischemic insults exerted a primary effect on nearly all response measurements. We also evaluated the data for insult interactions and identified significant interactions between ischemic insults and pressure. Altogether, findings indicate that pressure may sub-lethally effect cells and alter cellular metabolism (LDH_i) and membrane properties. Results suggest that renal ischemia may be the primary, but not the sole, cause of cellular injury induced by obstructive uropathy.     

Metabolism of canine kidneys in anaerobic ischemia and in aerobic ischemia by persufflation with gaseous oxygen

Summary The changes in the status of the adenylic acid-creatine phosphate system and in glycogen, glucose, and lactate were investigated in canine kidneys during preservation in anaerobic and aerobic ischemia at normo- and hypothermia. For anaerobic ischemia, the kidneys were stored without additional measures; for aerobic ischemia, the ischemic kidneys were persufflated with gaseous O₂ or gas mixtures containing O₂ via the renal artery or vein (orthograde and retrograde aerobic ischemia respectively). Prior to preservation, the kidneys were flushed with cell-free perfusates.Changes in the metabolic status characteristic of anaerobic ischemia were a decrease in the tissue levels of ATP, creatine phosphate, glycogen, glucose, and the sum of adenine nucleotides, a transitory increase in ADP and AMP, and an increase in the tissue level of lactate. Hypothermia caused a retardation of the anaerobic alterations; the loss of ATP was slowed down by factors of 2 and 9.5 when the temperatures were lowered from 37° to 26° and 6°C, respectively.In aerobic ischemia with orthograde and retrograde persufflation of 100% O₂ (persufflation pressure = PP 60 and 30 mm Hg), deviations from a regular metabolic status developed very slowly. While the ATP-level decreased to 50% of the control value within scarcely 30 min of anaerobic ischemia at 6°C, it was almost 48 h before a similar loss of ATP occurred during orthograde aerobic ischemia at 6°C. Reduction of O₂ in the persufflation gas to 40 and 21 vol-% resulted in less adequate preservation of the metabolic status. Increasing PP to 100 mm Hg for orthograde persufflation had no effect. An increase in PP to 60 mm Hg for retrograde persufflation resulted in a further delay in the metabolic deterioration. The long-term preservation of a regular metabolic status or the slow deviation of the metabolic pattern from the normal range during ischemia with O₂-persufflation indicates a significantly smaller energy deficit than during storage in anaerobic ischemia. It must be attributed to a sufficient aerobiosis in the aerobically ischemic kidney, during which organogenic substrates brought in by the initial perfusion are utilized.Supported by a grant from the Deutsche Forschungsgemeinschaft in the Sonderforschungsbereich 68.     

Heme oxygenase–carbon monoxide pathway is involved in regulation of respiration in medullary slice of neonatal rats

Carbon monoxide (CO) is a novel biological messenger molecule. It is well known that CO can be synthesized in mammalian cells. In addition, CO is also demonstrated to participate in many physiological processes, such as vasomotion, thermoregulation and respiratory regulation. The purpose of our present study was to investigate the role of heme oxygenase–carbon monoxide (HO–CO) pathway in central regulation of respiration. The experiments were carried out on the medullary slices of neonatal Sprague–Dawley rats. The discharge activity of the hypoglossal rootlets was recorded to indicate the central rhythmic respiratory activity and its duration (DD), interval (DI), frequency (DF) and integrated amplitude (IA) were analyzed. The slices were perfused with ZnPP-9 (a potent inhibitor of heme oxygenase), CO and hemin (substrate of heme oxygenase), respectively, to observe their effects on respiratory activity. The results obtained were as follows: ZnPP-9 could decrease DD, DI and IA, and increase DF (P < 0.05); exogenous CO caused a decrease in DD and DF, and an increase in DI and IA (P < 0.05); in response to hemin, DI and IA decreased, DF increased (P < 0.05), and DD did not change significantly (P > 0.05); administration of both ZnPP-9 and hemin could decrease DI, and increase DF (P < 0.05), but did not affect DD and IA significantly (P > 0.05). It can be concluded from the results above that the HO–CO pathway may be involved in the regulation of rhythmic respiration at the level of medulla oblongata.     

In vitro evidence of possible influence of scutellarein towards bile acids' metabolism

Background

The glucuronidation process has been regarded as the key elimination process for toxic bile acids. UDP-glucuronosyltransferase (UGT) 1A3 is one important metabolizing enzyme involved in this process.

Objective

To evaluate the inhibition of UGT1A3 by scutellarein which is an important herbal ingredient using in vitro method, trying to indicate the possibility of toxicity due to the accumulation of toxic bile acids.

Methods

Due to the difficulty to gain the standards of biles acids'' glucuronides, the recombinant UGT1A3-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction was employed to profile the activity of UGT1A3.

Results

The results showed that scutellarein inhibited UGT1A3 in a concentration-dependent behaviour. Competitive inhibition was demonstrated using both Dixon plot and Lineweaver-Burk plot, and the inhibition kinetic parameter (K_i) was calculated to be 5.8uM.

Conclusion

All these data reminded the necessary monitoring of the levels of bile acids in plasma when utilizing scutellarein and the herbs containing this compound.     

In vitro production of an active neurotrophic factor, neuregulin-1: qualitative comparison of different cell-free translation systems

The individual biological activities of many neurotrophic factors and their variants, which are produced by alternative splicing and proteolytic processing, often remain to be characterized. Bacterial protein production combined with protein refolding and purification is a conventional procedure to obtain active neurotrophic factors; however, the procedure is time consuming and appropriate protein refolding in vitro is sometimes unpredictable. Here we examined three distinct cell-free translation systems: reticulocyte lysate, Hela cell lysate and wheat germ extract, which may allow us to produce biologically active factors in a single tube. Taking type-I neuregulin-1 beta3 as an example, we produced neuregulin-1 protein from its mRNAs flanked by Cap nucleotide and/or internal ribosome entry site (IRES) and compared the yields and biological activity of translation products from these systems. The protein yield from IRES+ mRNA was highest in the Hela cell-free system, while background translation was lowest in the wheat germ system. The biological activity of both translation products was modest or negligible, however. Neuregulin-1 protein was produced in reticulocyte lysate at yields of 19 pmol/mL (~500 ng/mL); furthermore, it was potent at phosphorylating ErbB4 receptor and able to bind to heparin sulfate. These results demonstrate that the reticulocyte lysate translation system produces active neurotrophic factors in vitro and is useful for radiolabeling or preliminary assessment of novel neurotrophic factors and their variants.     

Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

Although the metabolism of early bovine embryos has not been fully elucidated,several publications have addressed this important issue to improve cultureconditions for cattle reproductive biotechnologies, with the ultimate goal ofproducing in vitro embryos similar in quality to those developingin vivo. Here, we review general aspects of bovine embryometabolism in vitro and in vivo, and discuss theuse of metabolic analysis of embryos produced in vitro to assessviability and predict a viable pregnancy after transference to the female tract.     

Protein expression by Aeromonas hydrophila during growth in vitro and in vivo

Expression of Aeromonas hydrophila cellular and extracellular products (ECPs) was examined following culture of the bacterium in vitro, in Tryptic Soy Broth (TSB), and in vivo, in dialysis tubing placed within the peritoneal cavity of common carp (Cyprinus carpio L.). Whole cell (WC), outer membrane proteins (OMPs) and ECP components of the bacteria were analysed by 1 dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE). Additionally, 2D SDS-PAGE was used to analyse WC preparations. The aim of the study was to identify unique and common proteins up-regulated in vivo. Unique bands were seen in the 1D gels at 58 and 55 kDa for WC and OMP preparations, respectively, for all the four virulent and two avirulent isolates cultured in vivo. Bands of increased intensity were also observed at 70, 55, 50 and 25 kDa with WC preparations for all virulent isolates cultured in vivo. Analysis of WC by 2D SDS-PAGE revealed that bacteria cultured in vivo expressed a number of unique spots, mostly between 30 and 80 kDa with pI values ranging from 5.0 to 6.0. The unique proteins identified in vivo may be involved in the virulence of the bacterium and their potential as vaccine candidates is currently being investigated.     

肠缺血再灌注后应用抗组胺药对大鼠生存率的影响

目的： 在肠缺血再灌注后静脉给予抗组胺药酮替芬,观察它对SD大鼠小肠功能结构以及存活率的影响。方法： 72只SD大鼠随机分为3组：S组（假手术组）、M组（模型组）、K组（缺血再灌注＋酮替芬1 mg/kg组）,每组又分术后3 d、7 d两亚组。复制小肠缺血/再灌注模型,K组在再灌注前5 min和再灌注后3 d静脉注射酮替芬。观察各组大鼠3 d（S3,M3,K3）、7 d（S7,M7,K7）的存活率、存活状态,检测小肠组织组胺浓度,观察肠黏膜病理结构变化和肠黏膜肥大细胞（intestinal mucosal mast cell,IMMC）超微结构及类胰蛋白酶表达比较。结果： M3组、K3组存活率低于S3组（P<0.05）; M7组存活率低于S7组（P<0.05）,K3组与M3组、K7组与S7组存活率差别无显著（P>0.05）。K7组存活率高于M7组（P<0.05）。M组、K组肠黏膜绒毛损伤轻微。电镜示M组微绒毛肿胀脱落,K组微绒毛不齐;S组IMMC超微结构正常,M组脱颗粒现象明显,K组次之。各组间肠黏膜Chiu’s评分、IMMC计数及类胰蛋白酶表达比较均无显著差异（P>0.05）。K7组肠组织组胺浓度低于S7组和M7组（P<0.05）。 结论： 肠缺血再灌注后应用抗组胺药酮替芬能改善大鼠小肠黏膜结构并增加大鼠生存率,IMMC活化脱颗粒组胺释放可能不利于肠缺血再灌注损伤大鼠的长期生存。     

Inhibition of brain creatine kinase activity after renal ischemia is attenuated by N-acetylcysteine and deferoxamine administration

Encephalopathy may accompany acute or chronic renal failure, and the mechanisms responsible for neurological complications in patients with renal failure are poorly known. Considering that creatine kinase (CK) is important for brain energy homeostasis and is inhibited by free radicals, and that oxidative stress is probably involved in the pathogenesis of uremic encephalopathy, we measured CK activity (hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex) in brain if rats submitted to renal ischemia and the effect of administration of antioxidants (N-acetylcysteine, NAC and deferoxamine, DFX) on this enzyme. We verified that CK activity was not altered in cerebellum and striatum of rats. CK activity was inhibited in prefrontal cortex and hippocampus of rats 12h after renal ischemia. The treatment with antioxidants prevented such effect. Cerebral cortex was also affected, but in this area CK activity was inhibited 6 and 12h after renal ischemia. Moreover, only NAC or NAC plus DFX were able to prevent the inhibition on the enzyme. Although it is difficult to extrapolate our findings to the human condition, the inhibition of brain CK activity after renal failure may be associated to neuronal loss and may be involved in the pathogenesis of uremic encephalopathy.     

The crucial amino acid residue related to inactivation of Vibrio vulnificus hemolysin

Vibrio vulnificus, an opportunistic human pathogen causing fetal septicemia, produces a 50-kDa pore-forming toxin as a virulence factor. This toxin consists of 451 amino acid residues; however, there are two types of this toxin on the basis of the difference of some amino acid residues, type 1 (Leu(281), Ser(415), Asn(435)/Asp(435), Asn(438)) and type 2 (Ile(281), Asn(415), Asn(435), Thr(438)). In the present study, two characteristic properties of type 2 toxin that was elaborated by V. vulnificus cells or synthesized by the in vitro system were compared to those of type 1 toxin. Type 2 toxin was found to be more resistant to spontaneous inactivation at 37 degrees C and to specific inactivation by cholesterol. On the other hand, a variant of type 2 toxin (Asp(435), Asn(438)) showed the same properties as type 1 toxin. The replacement of the 438th Asn to Thr (N438T), but not the 435th Asp to Asn (D435N), resulted in reversion of the variant type 2 toxin to typical type 2 toxin. These findings indicate that a single amino acid residue, Thr(438), may be critical for higher stability of type 2 toxin.     

Oocyte maturity in relation to woman's age in in vitro fertilization cycles stimulated by single regimen

Purpose

During stimulated in vitro fertilization (IVF) cycle, up to 30% of the recovered oocytes are immature ones which have poor fertilization capacity; however, the precise influencing factors are largely unknown. Here, we analyzed the association of oocyte immaturity with woman''s age in IVF cycles stimulated by single regimen.

Materials and Methods

A total of one-hundred ninety five IVF cycles stimulated by recombinant FSH and GnRH antagonist protocol between 2003 and 2009 were analyzed retrospectively. The mean age of women was 34.2±4.0 (26-45 years). After triggering by exogenous hCG, an ultrasound-guided retrieval of oocytes was performed 35-36 hours later. All clinical data were stratified by woman''s age; group I: ≤30 (n=36), II: 31-35 (n=83), III: 36-40 (n=57), and IV: ≥41 (n=19).

Results

The total retrieved oocytes, as well as immature oocytes, were significantly lower in group IV, however, the mean % of immature oocytes was significantly higher in group IV than other age groups. Oocyte immaturity tended to decrease as increasing age in women aged 40 years or less.

Conclusion

In stimulated IVF cycle, much higher oocyte immaturity was noted in women aged 41 years or more.     

Evidence against a role of gap junctions in vestibular compensation

Vestibular compensation following unilateral labyrinthectomy is associated with modifications of the membrane and firing properties of central vestibular neurons. To determine whether gap junctions could be involved in this process, immunofluorescent detection of neuronal connexin 36 and astrocytic connexin 43 was performed in the medial vestibular nucleus (MVN) of rats. In non-lesioned animals, strong staining was observed with anti-connexin 43 antibodies, while moderate staining was obtained with the anti-connexin 36 antibody. However, the expression of either type of connexin was not modified following unilateral labyrinthectomy. These morphological observations were complemented by pharmacological tests performed during extracellular recordings of MVN neurons in guinea pig brainstem slices. In non-lesioned animals, the gap junction blocker carbenoxolone reversibly decreased or suppressed the spontaneous discharge of about 60% of MVN neurons. This reduction was often associated with a long-duration disruption of the regularity of spike discharge. Both effects were mimicked by several other gap junction blockers, but not by glycyrrhizic acid, an analog of carbenoxolone that does not block gap junctions but reproduces its non-specific effects, nor by the selective inhibitor of astrocytic connexin-based networks endothelin-1. Similar effects of carbenoxolone were obtained on the spontaneous activity of ipsilesional MVN neurons recorded in brainstem slices taken from labyrinthectomized animals. Altogether, these results suggest that neuronal gap junctions are involved in shaping the spontaneous activity of MVN neurons. However, unilateral labyrinthectomy does not affect the expression of gap junctions in vestibular nuclei nor their implication in the regulation of neuronal activity.     

Effects of Urtica dioica on hepatic ischemia-reperfusion injury in rats

OBJECTIVES:

To evaluate the effects of Urtica dioica on hepatic ischemia‐reperfusion injury.

METHODS:

Thirty adult male Wistar albino rats were divided into three groups: sham group (group 1), control group (group 2), and Urtica dioica group (group 3). All the rats were exposed to hepatic ischemia for 60 min, followed by 60 min of reperfusion. In group 2, a total of 2 ml/kg 0.9% saline solution was given intraperitoneally. In group 3, a total of 2 ml/kg Urtica dioica was given intraperitoneally. At the end of the procedure, liver tissue and blood samples were taken from all rats. Serum aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, ceruloplasmin, catalase, paraoxonase, arylesterase, and lipid hydroperoxide levels were measured. Liver tissue histopathologies were also evaluated by light microscopy.

RESULTS:

Serum aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase levels were significantly higher in group 2 than in group 1, and significantly lower in group 3 than in group 2. Also, group 2 had higher serum lipid hydroperoxides and ceruloplasmin levels but lower catalase, paraoxonase, and arylesterase levels than group 1.In group 3, serum lipid hydroperoxides and ceruloplasmin levels were significantly lower, and catalase, paraoxonase, and arylesterase levels were higher than those in group 2. Histopathological examination showed that liver tissue damage was significantly decreased in group 3 compared with group 2.

CONCLUSIONS:

Urtica dioica has a protective effect on the liver in hepatic ischemia‐reperfusion‐injured rats.     

Metabolomic profiling of human follicular fluid from patients with repeated failure of in vitro fertilization using gas chromatography/mass spectrometry

Objective: To establish a gas chromatography/mass spectrometry (GC/MS)-based metabolomics method to compare the metabolites in the follicular fluid (FF) from patients with in vitro fertilization (IVF) and repeated IVF failure (RIF). Methods: A prospective study was employed in Center for Reprodutive Medcine, Renji Hospital, Shanghai, China, between January and October 2010. FF samples were collected from 13 patients with RIF and 15 patients who achieved pregnancy after the first IVF cycle. Results: Partial least squares (PLS) discriminant analysis of the PCA data revealed that the samples were scattered into two different regions. FF from the two groups differed with respect to 20 metabolites. FF from RIF group showed elevated levels of several amino acids (valine, threonine, isoleucine, cysteine, serine, proline, alanine, phenylalanine, lysine, methionine and ornithine), and reduced levels of dicarboxylic acids, cholesterol and some organic acids. Conclusions: The studies corroborated successful determination of the levels of metabolite in the FF.     

Progress in the development of shrimp cell cultures in Thailand

Primary shrimp cell cultures were developed from lymphoid organ and ovaries of black tiger shrimp, Penaeus monodon, in double-strength Leibovitz's L-15 medium supplemented with 15% fetal bovine serum, 1% glucose, 5 g/L NaCl, 15% shrimp meat extract. The optimum conditions for primary culture in vitro were obtained in L-15 medium with an osmolality of approximately 730 ± 10 mmol/kg, a temperature range of 25--28 °C and incubation in a normal atmosphere. However, basal medium supplemented with 0.01% cholesterol could enhance good growth and cells performance initiated from lymphoid organ. Both epithelial-like and fibroblastic-like cells were observed from those organs within 2 days incubation. Within 3 days, 80% confluent monolayers were obtained from the lymphoid organ while cultures from other tissues required 5 days. Cultures were maintained for at least 43 days. Only cells from lymphoid organ could be subcultured and confluent monolayers achieved within 10 days post-spilt. Healthy cultures of the lymphoid cells did not persist beyond the third passage. Application of these primary shrimp cell cultures for studying pathogenic viruses of shrimp in vitro will be discussed.     

Up-regulation of P2X7 receptor-immunoreactivity by in vitro ischemia on the plasma membrane of cultured rat cortical neurons

Mixed neuronal/astrocytic cortical cell cultures of the rat were incubated for 2 or 12 h under normoxic or ischemic conditions. Subsequent flow cytometric analysis with an anti-P2X₇ receptor antibody directed against an extracellular epitope indicated the up-regulation of these receptors at the plasma membrane by 12 h of ischemia. Labelling of MAP-2 immunopositive neurons by an anti-P2X₇ antibody directed against a C-terminal epitope, documented the selectivity of the ischemia-induced increase in receptor-density for the neuronal population. By contrast, staining of GFAP immunopositive astrocytes by the same anti-P2X₇ antibody excluded any effect of ischemia on the astrocytic density of P2X₇ receptors. The ischemic up-regulation of neuronal P2X₇ receptors is in perfect agreement with the previously reported facilitation of transmitter release from the GABAergic non-pyramidal cell type in such cultures [K. Wirkner, A. Köfalvi, W. Fischer, A. Günther, H. Franke, H. Gröger-Arndt, W. Nörenberg, E. Madarasz, E.S. Vizi, D. Schneider, B. Sperlagh, P. Illes, Supersensitivity of P2X₇ receptors in cerebrocortical cell cultures after in vitro ischemia, J. Neurochem. 95 (2005) 1421–1437].     

Poly (ADP) ribose synthetase inhibition in alveolar macrophages undergoing hypoxia and reoxygenation

BACKGROUND: Inhibition of the nuclear enzyme poly ribose synthetase (PARS) protects against in vivo lung ischemia reperfusion injury (LIRI). The effectiveness of intratracheal treatment suggests that PARS inhibition may primarily modulate alveolar macrophage (AM) activation. These studies attempted to characterize the effects of PARS on AM activation in response to oxidative stress. METHODS: Primary cultures of AM were rendered hypoxic for 2 h and reoxygenated for up to 4 h. Cells were preincubated with INO-1001, a specific PARS inhibitor 1 h prior to hypoxia. Gel shift assays characterized nuclear factor kappa B (NFkappaB), and enzyme linked immunosorbent assay quantitated chemokine/cytokine protein secretion. RESULTS: Hypoxia and reoxygenation resulted in an increase in the early nuclear translocation of NFkappaB, and an increase in the secretion of the cytokine tumor necrosis factor-alpha (TNF-alpha), chemokines macrophage inflammatory protein (MIP-1alpha), monocyte chemoattractant protein one (MCP-1) and cytokine induced neutrophil chemoattractant (CINC). Pretreatment of AM with INO-1001 decreased both the early translocation of NFkappaB and the production of TNF-alpha (p<0.05) and MIP-1alpha p=0.02, but did not affect CINC or MCP-1 production. CONCLUSIONS: These findings indicate that PARS inhibition in the AM blunts their response to oxidative stress and may help explain the protective effects of intratracheal PARS inhibition in LIRI.     

In vitro production of monoclonal antibodies to cultured embryonic chick limb mesenchyme

A simple, rapid protocol for the in vitro production of monoclonal antibodies (MAbs) that recognize native antigens in cultured chick limb mesenchyme during chondrogenic differentiation is described. Murine lymphocytes were stimulated by direct exposure to methanol-fixed micromass cultures of limb mesenchyme derived from the distal tip of stage 25 chick limb buds. Initial immunohistochemical characterization of two antibodies (DIDI and DIIA5) produced by this method showed preferential localization of reactivity with antigens in developing cartilage nodules during chondrogenesis in cultured chick limb mesenchyme. This study demonstrates the utility of in vitroimmunization of lymphocytes for the production of MAbs to native antigens expressed by differentiating embryonic limb cells in culture. Immunohistochemical data provided by DIDI and DIIA5 suggest that antigens bearing these epitopes may be important in early morphogenetic events during limb skeletal development.